Method for Increasing Expression Yield of a Protein of Interest

Abstract

ates to a method of producing a protein of interest comprising: (a) cultivating a mutant cell under conditions conducive for production of the protein wherein the mutant has a reduced or no expression of an endogenous polypeptide shown in SEQ ID NO: 2 compared to a parent host cell grown under the same conditions; and optionally (b) recovering the protein of interest.

Description

REFERENCE TO A SEQUENCE LISTING

[0001]This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002]1. Field of the Invention

[0003]The present invention relates to methods of increasing expression yield of a protein of interest. The present invention in particular relates to isolated polypeptides and isolated polynucleotides encoding polypeptides of the invention. The invention furthermore relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

[0004]2. Description of the Related Art

[0005]The use of recombinant host cells in the expression of heterologous proteins has in recent years greatly simplified the production of large quantities of commercially valuable proteins which otherwise are obtainable only by purification from their native sources. Currently, there is a varied selection of expression systems from which to choose for the production of any given protein, including bacterial and eukaryotic hosts. The selection of an appropriate expression system often depends not only on the ability of the host cell to produce adequate yields of the protein in an active state, but, to a large extent, may also be governed by the intended end use of the protein.

[0006]Typically improved yields have been obtained by improving the stability of the protein product by using different protease mutants or increasing the expression by using a strong promoter for controlling the expression or improving the secretion pathway by using the most efficient signal peptides.

[0007]Host cells used for heterologous protein expression are known to be in a state of stress since the protein synthsizing machinery will be pushed to the limit (Rutkowski, D. T., and Kaufman, R. J. (2004),"A trip to the ER: coping with stress", Trends in Cell Biology 14 (1): 20-28). Proteins involved in stress responses could therefore be advantageously manipulated leading to improved yield of protein products.

[0008]It is an object of the present invention to provide such polypeptides the expressions of which are induced or increased during stress conditions and provide methods for improving yield by modifying the expression.

SUMMARY OF THE INVENTION

[0009]The present invention relates to isolated polypeptides selected from the group consisting of: [0010](a) a polypeptide comprising an amino acid sequence having at least 66%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide of SEQ ID NO: 2; [0011](b) a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions, even more preferably at least medium-high stringency conditions, and most preferably at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) cDNA sequence contained in the DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii); [0012](c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide coding sequence of SEQ ID NO: 1; [0013](d) a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NO: 2.

[0014]The present invention also relates to isolated polynucleotides encoding the polypeptides of the invention.

[0015]The present invention also relates to nucleic acid constructs, recombinant expression vectors, recombinant host cells comprising the polynucleotides, and methods of producing a polypeptide of the invention.

[0016]The present invention in a further aspect relates to a method of producing the polypeptide of the invention, comprising: (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

[0017]The present invention in a further aspect relates to a method of producing the polypeptide of the invention, comprising: (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

[0018]The present invention also relates to a mutant fungal host, wherein the mutant has a reduced or no expression of an endogenous polypeptide selected from the group consisting of: [0019](a) a polypeptide comprising an amino acid sequence having at least 66%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide of SEQ ID NO: 2; [0020](b) a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions, even more preferably at least medium-high stringency conditions, and most preferably at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) cDNA sequence contained in the DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii); [0021](c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide coding sequence of SEQ ID NO: 1; [0022](d) a polypeptide induced during apoptosis and comprising the motif: EXXCGXXGXMXXDPXXLPEGVXXXXXRXCA; and [0023](e) a variant comprising a substitution, deletion, and/or insertion of one or several amino acids of the mature polypeptide of SEQ ID NO: 2.

[0024]The present invention also relates to a method of producing a protein of interest comprising: (a) cultivating a mutant cell under conditions conducive for production of the protein wherein the mutant has a reduced or no expression of an endogenous polypeptide compared to a parent host cell grown under the same conditions, and wherein the expression of said endogenous polypeptide commits the cell to apoptosis; and optionally (b) recovering the protein of interest.

[0025]The present invention also relates to methods of producing a protein of interest comprising: (a) cultivating the mutant cell according to the invention further expressing the protein of interest under conditions conducive for production of the protein; and optionally (b) recovering the protein.

BRIEF DESCRIPTION OF THE FIGURES

[0026]FIG. 1 shows the cDNA sequence and the deduced amino acid sequence of Aspergillus oryzae ZY082808 (SEQ ID NOs: 1 and 2, respectively).

DEFINITIONS

[0027]Polypeptide induced during apoptosis: The term "expression of said endogenous polypeptide commits the cell to apoptosis" is defined herein to mean that once the endogenous polypeptide has been expressed the host cell will eventually undergo apoptosis.

[0028]Apoptosis: The term "apoptosis" is defined herein as cell death resulting from induction of an internal suicide program which prior to the cell death includes cleavage of caspase substrates.

[0029]Isolated polypeptide: The term "isolated polypeptide" as used herein refers to a polypeptide that is isolated from a source. In a preferred aspect, the polypeptide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by SDS-PAGE.

[0030]Substantially pure polypeptide: The term "substantially pure polypeptide" denotes herein a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99%, most preferably at least 99.5% pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form, i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated. This can be accomplished, for example, by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

[0031]Mature polypeptide: The term "mature polypeptide" is defined herein as a polypeptide that is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In a preferred aspect, the mature polypeptide is amino acids 20 to 79 of SEQ ID NO: 2 based on the SignalP3.0 program that predicts amino acids 1 to 19 of SEQ ID NO: 2 are a signal peptide.

[0032]Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" is defined herein as a nucleotide sequence that encodes a mature polypeptide. In a preferred aspect, the mature polypeptide coding sequence is nucleotides 58 to 237 of SEQ ID NO: 1 based on the SignalP3.0 program that predicts nucleotides 1 to 57 of SEQ ID NO: 1 encode a signal peptide.

[0033]Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

[0034]For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the--nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment-Total Number of Gaps in Alignment)

[0035]For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the--nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment-Total Number of Gaps in Alignment)

[0036]Homologous sequence: The term "homologous sequence" is defined herein as a predicted protein that gives an E value (or expectancy score) of less than 0.001 in a tfasty search (Pearson, W. R., 1999, in Bioinformatics Methods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219) with the Aspergillus oryzae ZY082808 polypeptide of SEQ ID NO: 2.

[0037]Polypeptide fragment: The term "polypeptide fragment" is defined herein as a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of the mature polypeptide of SEQ ID NO: 2; wherein the fragment has retained its activity.

[0038]Subsequence: The term "subsequence" is defined herein as a nucleotide sequence having one or more (several) nucleotides deleted from the 5' and/or 3' end of the mature polypeptide coding sequence of SEQ ID NO: 1; wherein the subsequence encodes a polypeptide fragment having retained its activity.

[0039]Allelic variant: The term "allelic variant" denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0040]Isolated polynucleotide: The term "isolated polynucleotide" as used herein refers to a polynucleotide that is isolated from a source. In a preferred aspect, the polynucleotide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by agarose electrophoresis.

[0041]Substantially pure polynucleotide: The term "substantially pure polynucleotide" as used herein refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered protein production systems. Thus, a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polynucleotide material with which it is natively or recombinantly associated. A substantially pure polynucleotide may, however, include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, even more preferably at least 98% pure, most preferably at least 99%, and even most preferably at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form, i.e., that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively or recombinantly associated. The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

[0042]Coding sequence: When used herein the term "coding sequence" means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant nucleotide sequence.

[0043]cDNA: The term "cDNA" is defined herein as a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that are usually present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps before appearing as mature spliced mRNA. These steps include the removal of intron sequences by a process called splicing. cDNA derived from mRNA lacks, therefore, any intron sequences.

[0044]Nucleic acid construct: The term "nucleic acid construct" as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.

[0045]Control sequences: The term "control sequences" is defined herein to include all components necessary for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.

[0046]Operably linked: The term "operably linked" denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.

[0047]Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0048]Expression vector: The term "expression vector" is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that provide for its expression.

[0049]Host cell: The term "host cell", as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.

[0050]Modification: The term "modification" means herein any chemical modification of the polypeptide consisting of the mature polypeptide of SEQ ID NO: 2; or a homologous sequence thereof; as well as genetic manipulation of the DNA encoding such a polypeptide. The modification can be a substitution, a deletion and/or an insertion of one or more (several) amino acids as well as replacements of one or more (several) amino acid side chains.

DETAILED DESCRIPTION OF THE INVENTION

[0051]When fungi are used as expression host cells for the production of proteins the protein of interest should be produced in a sufficient amount. As discussed briefly above in order to improve yield of such proteins several factors have traditionally been studied. Host cells used in the production of such proteins, which could e.g. be a heterologous protein expressed recombinantly in the host or an endogenous protein produced in high quantities, will be in a stress situation (Rutkowski, D. T., and Kaufman, R. J. (2004),"A trip to the ER: coping with stress", Trends in Cell Biology 14 (1): 20-28). In the present invention expression yield have been improved by affecting the expression of specific factors the expression of which seems to be influenced by the level of stress the host cell is subjected to. Such factors have been identified using a cDNA library of A. oryzae expressed sequence tags (ESTs) in a DNA array. A number of genes were differentially regulated and among these the polypeptide according to the present invention.

Polypeptides of the Invention

[0052]In a first aspect, the present invention relates to isolated polypeptides comprising an amino acid sequence having a degree of identity to the mature polypeptide of SEQ ID NO: 2 of preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides have an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO: 2.

[0053]A polypeptide of the present invention preferably comprises the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises amino acids 20 to 79 of SEQ ID NO: 2, or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide comprises amino acids 20 to 79 of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of amino acids 20 to 79 of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide consists of amino acids 20 to 79 of SEQ ID NO: 2.

[0054]In a second aspect, the present invention relates to isolated polypeptides that are encoded by polynucleotides that hybridize under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained in SEQ ID NO: 1, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 1 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. In a preferred aspect, the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NO: 1.

[0055]The nucleotide sequence of SEQ ID NO: 1; or a subsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2; or a fragment thereof; may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length. For example, the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention.

[0056]A genomic DNA or cDNA library prepared from such other strains may, therefore, be screened for DNA that hybridizes with the probes described above and encodes a functionally homologous polypeptide. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NO: 1; or a subsequence thereof; the carrier material is preferably used in a Southern blot.

[0057]For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the mature polypeptide coding sequence of SEQ ID NO: 1; the cDNA sequence contained in SEQ ID NO: 1; its full-length complementary strand; or a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film.

[0058]In a preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1. In another preferred aspect, the nucleic acid probe is nucleotides 58 to 237 of SEQ ID NO: 1. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1.

[0059]For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.

[0060]For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS preferably at 45° C. (very low stringency), more preferably at 50° C. (low stringency), more preferably at 55° C. (medium stringency), more preferably at 60° C. (medium-high stringency), even more preferably at 65° C. (high stringency), and most preferably at 70° C. (very high stringency).

[0061]For short probes that are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5° C. to about 10° C. below the calculated T_m using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally.

[0062]For short probes that are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6× SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6× SSC at 5° C. to 10° C. below the calculated T_m.

[0063]In a third aspect, the present invention relates to isolated polypeptides encoded by polynucleotides comprising or consisting of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 1 of preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably 96%, 97%, 98%, or 99%, which encode an active polypeptide. See polynucleotide section herein.

[0064]In another aspect, the the isolated polypeptides comprise the following motif:

[0065]EXXCGXXGXMXXDPXXLPEGVXXXXXRXCA,

[0066]wherein x is any amino acid. In the above motif, the accepted IUPAC single letter amino acid abbreviation is employed.

[0067]In a still further aspect, the present invention relates to artificial variants comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of SEQ ID NO: 2; or a homologous polypeptide thereof. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

[0068]Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, LeuNal, Ala/Glu, and Asp/Gly.

[0069]In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be substituted for amino acid residues of a wild-type polypeptide. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

[0070]Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

[0071]Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to a polypeptide according to the invention.

[0072]Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127). Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

[0073]The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 2, such as amino acids 20 to 79 of SEQ ID NO: 2, is 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably 5, more preferably 4, even more preferably 3, most preferably 2, and even most preferably 1.

Sources of Polypeptides

[0074]A polypeptide of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a nucleotide sequence is produced by the source or by a strain in which the nucleotide sequence from the source has been inserted. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0075]A polypeptide of the present invention the expression of which commits the host cell to apoptosis may be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, lrpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide.

[0076]In a preferred aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide.

[0077]In another preferred aspect, the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma Iongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

[0078]In another preferred aspect, the polypeptide is an Aspergillus oryzae, Aspergillus niger, polypeptide.

[0079]In a more preferred aspect, the polypeptide is an Aspergillus oryzae polypeptide. In a most preferred aspect, the polypeptide is an Aspergillus oryzae A1560 (NBRC4177) polypeptide, e.g., the polypeptide comprising the mature polypeptide of SEQ ID NO: 2.

[0080]It will be understood that for the aforementioned species the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0081]Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0082]Furthermore, such polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The polynucleotide may then be obtained by similarly screening a genomic or cDNA library of such a microorganism. Once a polynucleotide sequence encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are well known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

[0083]Polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.

[0084]A fusion polypeptide can further comprise a cleavage site. Upon secretion of the fusion protein, the site is cleaved releasing the polypeptide of the invention from the fusion protein. Examples of cleavage sites include, but are not limited to, a Kex2 site that encodes the dipeptide Lys-Arg (Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-76; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu or Asp)-Gly-Arg site, which is cleaved by a Factor Xa protease after the arginine residue (Eaton et al., 1986, Biochem. 25: 505-512); a Asp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after the lysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); a His-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I (Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombin after the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); a Glu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease after the Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro site, which is cleaved by a genetically engineered form of human rhinovirus 3C protease after the Gln (Stevens, 2003, supra).

Polynucleotides

[0085]The present invention also relates to isolated polynucleotides comprising or consisting of nucleotide sequences that encode polypeptides the expression of which commits the host cell to apoptosis of the present invention.

[0086]In a preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 1. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding sequence of SEQ ID NO: 1. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 58 to 237 of SEQ ID NO: 1. The present invention also encompasses nucleotide sequences that encode polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or the mature polypeptide thereof, which differ from SEQ ID NO: 1 or the mature polypeptide coding sequence thereof by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 1 that encode fragments of SEQ ID NO: 2.

[0087]The present invention also relates to mutant polynucleotides comprising or consisting of at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 1, in which the mutant nucleotide sequence encodes the mature polypeptide of SEQ ID NO: 2.

[0088]The techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used. The polynucleotides may be cloned from a strain of Aspergillus, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleotide sequence.

[0089]The present invention also relates to isolated polynucleotides comprising or consisting of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 1 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity, which encode an active polypeptide.

[0090]Modification of a nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., artificial variants that differ in specific activity, thermostability, pH optimum, or the like. The variant sequence may be constructed on the basis of the nucleotide sequence presented as the mature polypeptide coding sequence of SEQ ID NO: 1, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not give rise to another amino acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

[0091]It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by an isolated polynucleotide of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, supra). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for maintained activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, e.g., de Vos et al., 1992, supra; Smith et al., 1992, supra; Wlodaver et al., 1992, supra).

[0092]The present invention also relates to isolated polynucleotides encoding polypeptides of the present invention, which hybridize under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained in SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii); or allelic variants and subsequences thereof (Sambrook et al., 1989, supra), as defined herein. In a preferred aspect, the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NO: 1.

[0093]The present invention also relates to isolated polynucleotides obtained by (a) hybridizing a population of DNA under very low, low, medium, medium-high, high, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii); and (b) isolating the hybridizing polynucleotide, which encodes a polypeptide the expression of which commits the host cell to apoptosis. In a preferred aspect, the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NO: 1.

Nucleic Acid Constructs

[0094]The present invention also relates to nucleic acid constructs comprising an isolated polynucleotide of the present invention operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0095]An isolated polynucleotide encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art.

[0096]The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0097]Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase); and mutant, truncated, and hybrid promoters thereof.

[0098]In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

[0099]The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.

[0100]Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

[0101]Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

[0102]The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.

[0103]Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0104]Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

[0105]The control sequence may also be a polyadenylation sequence; a sequence operably linked to the 3' terminus of the nucleotide sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used in the present invention.

[0106]Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.

[0107]Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

[0108]The control sequence may also be a signal peptide coding sequence that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. The foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell of choice, i.e., secreted into a culture medium, may be used in the present invention.

[0109]Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, Humicola insolens endoglucanase V, and Humicola lanuginosa lipase.

[0110]Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0111]In one embodiment the signal peptide is the normal signal associated with the polypeptide of the invention.

[0112]In one aspect, the signal peptide comprises or consists of amino acids 1 to 19 of SEQ ID NO: 2. In another aspect, the signal peptide coding sequence comprises or consists of nucleotides 1 to 57 of SEQ ID NO: 1.

[0113]The control sequence may also be a propeptide coding sequence that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836).

[0114]Where both signal peptide and propeptide sequences are present at the amino terminus of a polypeptide, the propeptide sequence is positioned next to the amino terminus of a polypeptide and the signal peptide sequence is positioned next to the amino terminus of the propeptide sequence.

[0115]It may also be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, xyl and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence.

Expression Vectors

[0116]The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, a polynucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0117]The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0118]The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0119]The vectors of the present invention preferably contain one or more (several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0120]Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bargene of Streptomyces hygroscopicus.

[0121]The vectors of the present invention preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0122]For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0123]For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.

[0124]Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0125]Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

[0126]More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of the gene product. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0127]The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

Host Cells

[0128]The present invention also relates to recombinant host cells, comprising an isolated polynucleotide of the present invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

[0129]The host cell may be any eukaryote, such as a mammalian, insect, plant, or fungal cell.

[0130]In a preferred aspect, the host cell is a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).

[0131]In a more preferred aspect, the fungal host cell is a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0132]In an even more preferred aspect, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

[0133]In a most preferred aspect, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis cell. In another most preferred aspect, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred aspect, the yeast host cell is a Yarrowia lipolytica cell.

[0134]In another more preferred aspect, the fungal host cell is a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

[0135]In an even more preferred aspect, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

[0136]In a most preferred aspect, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred aspect, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In another most preferred aspect, the filamentous fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0137]Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

[0138]The present invention also relates to methods of producing a polypeptide of the present invention, comprising: (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. In a preferred aspect, the cell is of the genus Aspergillus. In a more preferred aspect, the cell is Aspergillus oryzae.

[0139]The present invention also relates to methods of producing a polypeptide of the present invention, comprising: (a) cultivating a recombinant host cell, as described herein, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

[0140]In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.

[0141]The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide as described herein.

[0142]The resulting polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0143]The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.

Removal or Reduction of ZY082808 Activity

[0144]The present invention also relates to a mutant of a parent cell, which comprises modifying, disrupting or deleting a polynucleotide sequence, or a portion thereof, encoding a polypeptide of the present invention, which results in the mutant cell producing less of the polypeptide than the parent cell when cultivated under the same conditions.

[0145]The mutant cell may be constructed by reducing or eliminating expression of a nucleotide sequence encoding a polypeptide of the present invention using methods well known in the art, for example, insertions, disruptions, replacements, or deletions. In a preferred aspect, the nucleotide sequence is modified so that the encoded polypeptide is inactivated. The nucleotide sequence to be modified may be, for example, the coding region or a part thereof essential for activity, or a regulatory element required for the expression of the coding region. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the nucleotide sequence. Other control sequences for possible modification include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, transcription terminator, and transcriptional activator.

[0146]Thus in one embodiment the present invention relates to a mutant fungal host, wherein the mutant has a reduced or no expression of an endogenous polypeptide selected from the group consisiting of:

[0147](a) a polypeptide comprising an amino acid sequence having at least 66%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide of SEQ ID NO: 2;

[0148](b) a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions, even more preferably at least medium-high stringency conditions, and most preferably at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) cDNA sequence contained in the DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii);

[0149](c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having [Edit accordingly] preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide coding sequence of SEQ ID NO: 1;

[0150](d) a polypeptide induced during apoptosis and comprising the motif: EXXCGXXGXMXXDPXXLPEGVXXXXXRXCA; and

[0151](e) a variant comprising a substitution, deletion, and/or insertion of one or several amino acids of the mature polypeptide of SEQ ID NO: 2.

[0152]In one further embodiment the endogenous polypeptide of the invention, the activity of which is reduced or eliminated in the mutant host, comprises the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises amino acids 20 to 79 of SEQ ID NO: 2, or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide comprises amino acids 20 to 79 of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of amino acids 20 to 79 of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide consists of amino acids 20 to 79 of SEQ ID NO: 2.

[0153]In a preferred aspect the mutant host cell is a filamentous fungal cell or a yeast cell. In a more preferred aspect the filamentous fungal host is selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

[0154]Preferably he Aspergillus cell is selected from Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae.

[0155]In another preferred embodiment the mutant filamentous fungal cell is an Aspergillus oryzae cell or an Aspergillus niger cell.

[0156]Modification or inactivation of the nucleotide sequence may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the nucleotide sequence has been reduced or eliminated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.

[0157]Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.

[0158]When such agents are used, the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and screening and/or selecting for mutant cells exhibiting reduced or no expression of the gene.

[0159]Modification of the nucleotide sequence may be accomplished by introduction, substitution, or removal of one or more (several) nucleotides in the gene or a regulatory element required for the transcription or translation thereof. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change in the open reading frame. Such modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. Although, in principle, the modification may be performed in vivo, i.e., directly on the cell expressing the nucleotide sequence to be modified, it is preferred that the modification be performed in vitro as exemplified below.

[0160]An example of a convenient way to eliminate or reduce expression of a nucleotide sequence by a cell is based on techniques of gene replacement, gene deletion, or gene disruption. For example, in the gene disruption method, a nucleic acid sequence corresponding to the endogenous nucleotide sequence is mutagenized in vitro to produce a defective nucleic acid sequence that is then transformed into the parent cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous nucleotide sequence. It may be desirable that the defective nucleotide sequence also encodes a marker that may be used for selection of transformants in which the nucleotide sequence has been modified or destroyed. In a particularly preferred aspect, the nucleotide sequence is disrupted with a selectable marker such as those described herein.

[0161]Alternatively, modification or inactivation of the nucleotide sequence may be performed by established anti-sense or RNAi techniques using a sequence complementary to the nucleotide sequence. More specifically, expression of the nucleotide sequence by a cell may be reduced or eliminated by introducing a sequence complementary to the nucleotide sequence of the gene that may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated.

[0162]The present invention further relates to a mutant cell of a parent cell that comprises a disruption or deletion of a nucleotide sequence encoding the polypeptide or a control sequence thereof, which results in the mutant cell producing less of the polypeptide or no polypeptide compared to the parent cell.

[0163]The polypeptide-deficient mutant cells so created are particularly useful as host cells for the expression of native and/or heterologous polypeptides. Therefore, the present invention further relates to a mutant cell according to the invention further comprising a gene encoding a native or heterologous protein and to methods of producing a native or heterologous polypeptide comprising: (a) cultivating the mutant cell under conditions conducive for production of the native or heterologous polypeptide; and (b) recovering the polypeptide. The term "heterologous polypeptides" is defined herein as polypeptides that are not native to the host cell, a native protein in which modifications have been made to alter the native sequence, or a native protein whose expression is quantitatively altered as a result of a manipulation of the host cell by recombinant DNA techniques.

[0164]In a further aspect, the present invention relates to a method of producing a protein of interest comprising: (a) cultivating a mutant cell under conditions conducive for production of the protein wherein the mutant has a reduced or no expression of an endogenous polypeptide compared to a parent host cell grown under the same conditions, and wherein the expression of said endogenous polypeptide commits the cell to apoptosis; and optionally (b) recovering the protein of interest.

[0165]In an even further aspect the present invention relatesto a method of producing a protein of interest, comprising: (a) cultivating the mutant cell having a reduced or no activity of the polypeptide of the invention under conditions conducive for production of the protein; and (b) recovering the protein. Particularly the protein is a heterologous protein.

[0166]The methods used for cultivation and purification of the product of interest may be performed by methods known in the art.

[0167]The methods of the present invention for producing a protein product are of particular interest in the production of eukaryotic polypeptides, in particular fungal proteins such as enzymes. The enzyme may be selected from, e.g., an amylolytic enzyme, lipolytic enzyme, proteolytic enzyme, cellulytic enzyme, oxidoreductase, or plant cell-wall degrading enzyme. Examples of such enzymes include an aminopeptidase, amylase, amyloglucosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, galactosidase, beta-galactosidase, glucoamylase, glucose oxidase, glucosidase, haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase, lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase, phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transferase, transglutaminase, or xylanase. The mutant cells may also be used to express heterologous proteins of pharmaceutical interest such as hormones, growth factors, receptors, and the like.

[0168]It will be understood that the term "eukaryotic polypeptides" includes not only native polypeptides, but also those polypeptides, e.g., enzymes, which have been modified by amino acid substitutions, deletions or additions, or other such modifications to enhance activity, thermostability, pH tolerance and the like.

Methods of Inhibiting Expression of a Polypeptide

[0169]In the production method according to the invention the reduction or elimination of the polypeptide according to the invention is particularly achieved by random mutation, site specific mutation, deletion, or by inhibiting the expression by administering a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of a polynucleotide of the present invention. In a preferred aspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.

[0170]The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA (miRNA). In a preferred aspect, the dsRNA is small interfering RNA (siRNAs) for inhibiting transcription. In another preferred aspect, the dsRNA is micro RNA (miRNAs) for inhibiting translation.

[0171]The present invention also relates to such double-stranded RNA (dsRNA) molecules, comprising a portion of the mature polypeptide coding sequence of SEQ ID NO: 1 for inhibiting expression of a polypeptide in a cell. While the present invention is not limited by any particular mechanism of action, the dsRNA can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to dsRNA, mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi).

[0172]Methods for making and using dsRNA molecules to selectively degrade RNA are well known in the art, see, for example, U.S. Pat. No. 6,506,559; U.S. Pat. No. 6,511,824; U.S. Pat. No. 6,515,109; and U.S. Pat. No. 6,489,127.

[0173]The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

Examples

[0174]Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Plasmids

[0175]pDV8 is described in patent WO2001068864, example 8. [0176]pJaL410 is described in patent WO2003008575, example 8. [0177]pJaL419 is described in patent WO2003008575, example 8. [0178]pJaL504 is described in example 1 and SEQ ID NO: 14. [0179]pJaL554 is described in patent WO2001068864, example 8. [0180]pJaL575 is described in example 1 and SEQ ID NO: 15. [0181]pJaL729 is described in patent WO2003008575, example 12. [0182]pJaL731 is described in example 1 and SEQ ID NO: 16. [0183]pJaL736 is described in example 1 and SEQ ID NO: 17. [0184]pJAL958 is described in example 1 and SEQ ID NO: 18. [0185]pMT2786 is described in example 2 and SEQ ID NO: 19. [0186]pMT3155 is described in example 2 and SEQ ID NO: 20. [0187]pMT3296 is described in example 2 and SEQ ID NO: 21. [0188]pMT3306 is described in example 2 and SEQ ID NO: 22. [0189]pMT3328 is described in example 3 and SEQ ID NO: 23. [0190]pMT3330 is described in example 3 and SEQ ID NO: 24. [0191]pMT3332 is described in example 3 and SEQ ID NO: 25. [0192]pMT3369 is described in example 4 and SEQ ID NO: 26. [0193]pMT3376 is described in example 4 and SEQ ID NO: 27. [0194]pMT3378 is described in example 4 and SEQ ID NO: 28. [0195]pMT3431 is described in example 4 and SEQ ID NO: 29.

Strains

[0195] [0196]ToC1512 is described in patent WO2005070962, example 11. [0197]JaL507 is described in example 1. [0198]BECh2 is described in WO 00/39322, example 1, which is further referring to JaL228 described in WO 98/12300, example 1.

Example 1

Identification of the ZY082808 Transcript

Construction of a Lipase Producing Strain of Aspergillus Oryzae

[0199]Construction of the Aspergillus Oryzae niaD Deletion Plasmid pJaL736

[0200]The single restriction endonuclease sites BamHIand BgIII in pDV8 were removed by two succeeding rounds of cutting with each of the restriction endonucleases and the free overhang-ends was filled in by treatment with Klenow polymerase and the four deoxyribonucleotides and ligated resulting in plasmid pJaL504 (SEQ ID NO: 14).

[0201]From pJaL504 a 2514 by fragment were amplified by PCR with primer 172450 and 172449 (SEQ ID NO: 3 and 4) and cloned into the vector pCR® 4Blunt-TOPO resulting in plasmid pJaL575 (SEQ ID NO: 15).

[0202]The repeat flanked pyrG selection marker from pJaL554 was moved as a 1998 by Asp718-HindIII fragment where the ends were completely filled in by treating with dNTP's and klenow polymerase. The fragment was cloned into completely filled in BssHII-Asp718 site of pJaL419 resulting in plasmid pJaL731 (SEQ ID NO: 16). The pyrG gene thereby replaces a 2200 by fragment encoding the 3' part of the niaD gene and the pyrG gene is then flanked by a 772 by fragment of the 5' end of niaD and a 698 by fragment of the 3' end of niaD. Finally, the deletion cassette of pJaL731 containing the two niaD flanks on either side of the pyrG selection marker was transferred as a 3500 by HindIII fragment into the HindIII site of the tk counter selectable plasmid pJaL575 to give the deletion plasmid pJaL736 (SEQ ID NO: 17). Note that pJaL736 contains a unique NotI site, which were used to linearize the plasmid prior to transformation into A. oryzae.

Construction of niaD Deleted A. Oryzae Strain, JaL507.

[0203]20 microgram of pJaL736 was cut with NotI and subsequently the enzyme was heat inactivated as recommended by the manufacturer (New England Biolabs). The plasmid was then ethanol precipitated and re-dissolved in Tris buffer (10 mM pH 8.0) at a concentration suitable for transformation into Aspergillus oryzae.

[0204]The linearized plasmid DNA was transformed into Aspergillus oryzae ToC1512 with selection for pyrG and counter selection of the tk gene on FDU plates as previously described in WO 0168864. Transformants were re-isolated twice and finally grown up in liquid medium (YPD). Transformants were tested for there ability to grow on nitrate as the sole nitrogen source. Chromosomal DNA was prepared as previously described in WO 0168864 form transformants that were not able to grow on nitrate which indicate that the niaD gene was disrupted and used for Southern analysis of the niaD locus with the aim to identify transformants in which a clean double cross-over between the chromosomal niaD and the deletion cassette had occurred. The chromosomal DNA was digested with BamHI. The Southern blot was first probed with the whole niaD gene excised as a 3430 by HindIII fragment from pJaL410. For the intact niaD locus, two bands of 2838 by and 1820 by respectively was appearing using this probe, while the niaD deleted derivative originating from the desired double cross-over resulted in a 4567 by band. The Southern was stripped of the first probe and re-probed with a probe excised as a 2059 by Asp718 fragment of pJaL410. For the intact niaD locus a 2838 by and a 1820 by BamHI band were appearing, while for the niaD deletion strain originating from the desired cross over no hybridization bands was obtained. A strain with the above characteristics was preserved as A. oryzae strain JaL507.

Construction of pJaL958

[0205]A variant of the Humicola lanuginosa lipase was cloned as a 901 by BamHI-SaII obtained from digestion of p960 (described in EP0305216 example 1 and FIG. 4) into pJaL729 digested with BamHI-XhoI resulting in pJaL958 (SEQ ID NO: 18).

Construction of A. Oryzae Strain JaL731

[0206]Plasmid pJaL958 was transformed into A. oryzae strain JaL507 and transformants were screened for lipase activity as described in WO2003008575, example 13-15. Among the transformants one displaying high lipase activity was selected and named JaL731.

TABLE-US-00001 Primer 172450 (SEQ ID NO: 3) 5'-GACGAATTCTCTAGAAGATCTCTCGAGGAGCTCAAGCTTCTGTA- CAGTGACCGGTGACTC Primer 172449 (SEQ ID NO: 4) 5'-GACGAATTCCGATGAATGTGTGTCCTG

[0207]Spores of JaL731 were subjected to mutagenesis using nitrosoguanidine (as described in Araujo, Ward and D'Souza. (1991). Biotechnology Techniques 5 (4): 283-288. Use of mutation strategies applied to Aspergillus terreus ATCC 52430 to obtain mutants with improved cellulase productivity). Mutants were screened for increased lipase expression in microtiter plates by using robotics, and a high yielding mutant, named 5-58, was selected. Lipase activity was measured as described by Haack et al., 2006 (Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation. Appl Microbiol Biotechnol 70: 482-487).

[0208]The two lipase producing strains, JaL731 and its high yield mutant 5-58, were compared by transcriptomic analysis of gene expression by using DNA microarray technique (Baldi and Hatfield, 2002 (DNA Microarrays and Gene Expression, Cambridge University Press, Cambridge); Knudsen, 2002 (A Biologist's Guide to Analysis of DNA microarray data. Wiley & sons, New York).

[0209]Spotted slides based on an inhouse cDNA library of A. oryzae expressed sequence tags were used. mRNA was extracted by the PolyATract® System 1000 (Promega Corporation, Madison, Wis.), starting with 1 g of cell material. cDNA synthesis and Cy3 and Cy5 labelling were performed with the aid of the CyScribe Post labelling Kit with CyScribe GFX purification kit (Amersham Biosciences, HiHerod, Denmark). Hybridization was performed using standard methods. Scanning of the slides was performed using a GMS 418 array scanner (Genetic MicroSystems Inc., Woburn, Mass.). The scanning images were quantified by using the ImaGene® 5 Software (Biodiscovery Inc., Marina Del Rey, Calif., USA). Statistical analysis of differential expression was performed using Genesight Software (Biodiscovery Inc., Marina Del Rey, Calif., USA).

[0210]Laboratory tank fermentation was carried out for each of the two strains JaL731 and 5-58, and sampling of biomass was done at two time points. Eight successful hybridizations were carried out between the two strains. The transcriptomic analysis showed differential regulation of the transcript ZY082808. In the high yield mutant, 5-58, the ZY082808 transcript was down-regulated in.

[0211]A number of genes were differentially regulated. However, also microarray data from analyses of other A. oryzae lipase strain pairs suggested ZY082808 as an interesting gene for follow up. Thus, gene expression of a high producing recombinant lipase strain Amsp173-40a was compared to an analogous lower producing recombinant lipase strain Amsp165-28a by using the DNA microarray technique. The strain Amsp173-40a is described by Haack et al. (2006) supra. Amsp165-28a was constructed in the same way as Amsp173-40a and selected as a low producing strain by test in laboratory tank fermentation. For Amsp173-40a increased swelling of the hyphal tip was observed in the fed batch phase with high lipase productivity and assumed secretion stress conditions as described and discussed by Haack et al. (2006) supra. For Amsp165-28a no swelling of the hyphal tip was observed. The transcriptomic analysis showed differential regulation of ZY082808 also in these strains, and ZY082808 was up-regulated in Amsp173-40a in a number of independent array experiments as shown in table 1. The reverse regulation of ZY082808 in the two different strain pairs suggested ZY082808 as a particular interesting gene for follow up.

TABLE-US-00002 TABLE 1 Amsp173-40a Amsp173-40a Amsp173-40a 5-58 Amsp165-28a Amsp165-28a Amsp165-28a JaL731 Exp1 Exp. 2 Exp. 3 Exp. 4 Fold Change P value Fold Change P value Fold Change P value Fold Change P value ZY 2.8 7.7 2.8 6.3 3.2 4.9 -3.1 < 082808 10^-6 10^-5 10^-5 0.001

Example 2

Expression Plasmids for Overexpression of the ZY082808 Encoded Peptide

Primers:

TABLE-US-00003 [0212]Primer 821 (SEQ ID NO: 5) 5'-TCTTGGTACCCTCAGCAAAGCCATCATTGC-3' Primer 822 (SEQ ID NO: 6) 5'-TCTCTCTAGACTCCCTCTGGTT- TAATACTCCG-3' Primer 829 (SEQ ID NO: 7) 5'-CATTTGGATCCACCATGAGACTCTCCC-3' Primer 841 (SEQ ID NO: 8) 5'-TTCTCTCGAGCTAAGGAAACCACCTCGGC-3'

[0213]The A. oryzae chromosomal sequence encoding the ZY082808 transcript was PCR amplified from A. oryzae strain BECh2 using primers 821 and 822 (SEQ ID 5 and 6) to give a 2.0 kb fragment. This fragment was cut at the Asp718 and Xba1 sites introduced by the applification primers 821 and 822. The ZY082808 encoding Asp718-Xba1 fragment was inserted in the vector pMT3155 (SEQ ID NO: 20) which had been cut with BsiW1 and Bln1. The pMT3155 vector contains markers to allow for selection of leucine prototrophy in E. coli and for selecting resistance to the herbicide BASTA in Aspergillus oryzae. The resulting plasmid capable of directing expression of the ZY082808 encoded peptide from its own original promoter was named pMT3296 (SEQ ID NO: 21).

[0214]To construct a plasmid expressing the ZY082808 encoded peptide from a strong tandem NA2/TPI promoter described in WO 2003008575, the ZY082808 ORF was PCR amplified from pMT3296 using primers 829 and 841 (SEQ ID NO: 7 and 8) to give a 0.25 kb fragment.

[0215]This fragment was cut at the BamH1 and Xho1 sites introduced by the PCR primers, and inserted in the vector pMT2786 (SEQ ID NO: 19) (which is a modified version of pMT2188 described in WO 2006/050737 example 2; in pMT2786 the URA3 marker has been replaced with the S. cerevisiae LEU2 marker which can be selected for in E. coli leuB mutants (leucine requiring), and in addition there is an approximately 2 kb "stuffer" inserted in pMT2786 between the BamH1 and Xho1 used for cloning) cut with Bam and Xho. The pMT2786 vector contains markers to allow for selection of leucine prototrophy in E. coli and for selecting for ability to grow on acetamide as sole nitrogen source in A. oryzae. The resulting expression plasmid was named pMT3306 (SEQ ID NO: 22).

[0216]pMT3296 was transformed into A. oryzae mutant strain 5-58 selecting for resistance to BASTA. 12 transformants were isolated and subsequently re-isolated twice through sporulation. Likewise, pMT3306 was transformed into strain 5-58 selecting for the ability to grow on acetamide as sole nitrogen source. No transformants were obtained growing detectably better than the background untransformed A. oryzae strain. The transformation experiment was repeated but again no transformants could be isolated.

[0217]The 12 transformants as well as the strain 5-58 were inoculated on Cove-N agar slants and incubated for one week at 34° C. For all isolates 500 ml shake flasks with 2 baffles and with 100 ml G1-Gly substrate was inoculated with 5 ml spore suspension and incubated over night at 30° C., 250 rpm. The G1-Gly substrate contained 18 g/l yeast extract, 24 g/l glycerol 87%, 1 ml/l pluronic and 5 g/l CaCO₃. Tapwater was used. pH was adjusted to 7.0 before sterilization for 20 min at 121° C. From each G1-Gly shake flask 5 ml was transferred to two 500 ml shake flasks with 2 baffles and with 100 ml 1/5MDU2B substrate. The 1/5MDU2B substrate contained 9.0 g/l maltose, 0.2 g/l MgSO₄.7H₂O, 0.2 g/l NaCl, 0.4 g/l K₂SO₄, 2.4 g/l KH₂PO₄, 0.1 ml/l trace metal, 0.02 ml/l pluronic, and 1.4 g/l yeast extract. Tapwater was used. The trace metal solution contained 14.2 g/l ZnSO4.7H₂O, 2.5 g/l CuSO4.5H₂O, 0.5 g/l NiCl2.6H₂O, 13.8 g/l FeSO4.7H₂O, 8.5 g/l MnSO4.H₂O and 3.0 g/l citric acid H₂O. pH was adjusted to 5.0 before sterilization for 20 min at 121° C. After sterilization 0.2 ml 50% urea was added pr. 100 ml substrate. The 1/5MDU2B shake flasks were incubated at 34° C., 270 rpm. Every shake flask was sampled after 2 days. Samples were centrifuged for 10 min at 3000 rpm. Supernatants are frozen at -20° C. Samples were thawed and lipase activity (LU/ml) was measured by using standard protocol. Two independent shake flaks experiments were carried out.

[0218]Four out of twelve transformants had reproducibly 2-3 times reduced lipase expression compared to 5-58 as shown in table 2. This indicates a negative effect of over-expression of ZY082808 as suggested by transcriptomic analysis. It can be seen that for e.g. transformant 5-58pMT3296#3 an increased expression was observed, which could be due to some random variation, i.e. copy number increase of the lipase gene combined with low expression of ZY082808 in that particular transformant. The results should be compared to transformation by the vector alone. Twenty pMT3155-transformants were fermented by using the same protocol as for the 5-58pMT3296 transformants.

[0219]No significant negative effect on lipase expression was observed for the twenty transformants as shown in table 3. This confirms the suggested negative effect of ZY082808 overexpression from the data shown in table 2.

TABLE-US-00004 TABLE 2 Experiment 1 Experiment 2 Lipase expression in lipase expression in Strain % of 5-58 % of 5-58 5-58pMT3296#1 126 109 5-58pMT3296#2 114 115 5-58pMT3296#3 138 140 5-58pMT3296#4 36 37 5-58pMT3296#5 27 33 5-58pMT3296#6 98 104 5-58pMT3296#7 33 38 5-58pMT3296#8 113 112 5-58pMT3296#9 144 119 5-58pMT3296#10 118 113 5-58pMT3296#11 93 87 5-58pMT3296#12 55 55 5-58 100 100

TABLE-US-00005 TABLE 3 Experiment 1 Experiment 2 lipase expression in lipase expression in Strain % of 5-58 % of 5-58 5-58 100 100 5-58pmt3155#1 74 98 5-58pmt3155#2 83 76 5-58pmt3155#3 73 114 5-58pmt3155#4 52 112 5-58pmt3155#5 75 102 5-58pmt3155#6 ND 95 5-58pmt3155#7 ND 129 5-58pmt3155#8 ND 110 5-58pmt3155#9 ND 101 5-58pmt3155#10 ND ND 5-58pmt3155#11 ND 96 5-58pmt3155#12 ND 106 5-58pmt3155#13 ND 124 5-58pmt3155#14 ND 126 5-58pmt3155#15 80 107 5-58pmt3155#16 94 102 5-58pmt3155#17 68 98 5-58pmt3155#18 70 104 5-58pmt3155#19 106 77 5-58pmt3155#20 78 105

Example 3

Construction of a Plasmid for Expression of a shRNA for ZY82808 Sequences

[0220]In order to have a visual marker by which to judge the degree of downregulation, a construct was made in which both ZY082808 and the wA gene needed for green spore development could be downregulated. Absence of the wA gene product leads to white spore color, and partial expression of the gene could be expected to lead to lighter shades of green than wt spore color.

Primers:

TABLE-US-00006 [0221] Primer 798 (SEQ ID NO: 9) 5'-TCTTGGATCCGTCGACCTTGGGGAAGTCATCACCG-3' Primer 868 (SEQ ID NO: 10) 5'-GTCATTAATCATTGAATTCTCGTCGCGACTCTC-3' Primer 865 (SEQ ID NO: 11) 5'-GTCGCGACGAGAATTCAATGATTAATGACCTTTTC- CTAAATATAC-3' Primer 867 (SEQ ID NO: 12) 5'-TCTTCTCGAGCCTAGGTATCCCAAAGTCAA- GTACGTCAGG-3' Primer 866 (SEQ ID NO: 13) 5'-TCTTCTCGAGCCTAGGCTCTCCCTCAACCTTCTACTG-3'

[0222]A fusion of the sequences encoding part of the wA (whiteA) product (Thomas et al., 1998, Microbiol and Mol Biol Reviews, 62(1): 35-54) and the ZY082808 sequence including part of the 5'UTR was made by SOE PCR: The wA sequences were amplified from A. oryzae strain BECh2 DNA using primers 798 and 868 (SEQ ID NO: 9 and 10, respectively) and fused to ZY082808 sequences amplified by primers 865 and 867 (SEQ ID NO: 11 and 12) from pMT3296 template.

[0223]The SOE fusion product was cut by BamH1 and Xho1 at the sites introduced by primers 798 and 867, respectively. The resulting approx. 1.2 kb fragment was ligated into BamH1-Xho1 cut vector pMT2786 to give plasmid pMT3328 (SEQ ID NO: 23).

[0224]A similar fusion of wA to a shorter piece of ZY082808 sequence (lacking 5'UTR) was made by fusing the wA fragment amplified by primers 798 and 868 to the ZY082808 sequence amplified by primers 865 and 866 (SEQ ID NO: 11 and SEQ ID NO: 13). The resulting approx. 1.0 kb fragment was cut by BamH1 and Xho1 at the sites introduced by primers 798 and 866, respectively. The Bam-Xho fragment was inserted in BamH1-Xho1 cut vector pMT2786 to give plasmid pMT3330 (SEQ ID NO: 24). Finally the Asp718-Xho1 (5.5 kb) and the Asp718-Bln1 (2.5 kb) fragments of pMT3328 were ligated to the Bln1-SaI1 (1.0 kb) fragment to give plasmid pMT3332 (SEQ ID NO: 25). pMT3332 can be selected in Aspergillus oryzae by the ability to grow on acetamide as the sole source of nitrogen. pMT3332 is constructed such that the strong NA2/TPI promoter drives the transcription of a shRNA containing sequences from both wA and ZY082808. Transformants with pMT3332 will thus contain double stranded RNA for both wA and ZY082808 and expression of both wA and ZY082808 is expected in various degrees to be knocked down in A. oryzae strains transformed with pMT3332.

[0225]pMT3332 was transformed into A. oryzae strain JaL731 and 24 transformants selected by their ability to grow on acetamide as sole source of nitrogen. Transformants were twice reisolated as described above.

[0226]The 24 transformants were evaluated in two independent shake flask experiments. As shown in table 4, five to ten of the transformants seemed to have 10-25% increased lipase expression compared to JaL731 although there is some uncertainty due to a high variation from experiment to experiment. This is in line with the clear negative effect of over-expression of ZY082808 in 5-58.

[0227]In summary both microarray data for various independent lipase strains, follow up studies by over-expression and by attempted down regulation by shRNA show, that ZY082808 has significant influence on lipase expression.

TABLE-US-00007 TABLE 4 Experiment 3 Experiment 4 Lipase expression Lipase expression in in Strain % of JaL731 % of JaL731 JaL731pMT3332#1 125 ND JaL731pMT3332#2 106 ND JaL731pMT3332#3 123 124 JaL731pMT3332#4 126 128 JaL731pMT3332#5 105 105 JaL731pMT3332#6 113 106 JaL731pMT3332#7 133 124 JaL731pMT3332#8 125 ND JaL731pMT3332#9 101 105 JaL731pMT3332#10 ND ND JaL731pMT3332#11 106 ND JaL731pMT3332#12 89 114 JaL731pMT3332#13 108 102 JaL731pMT3332#14 113 ND JaL731pMT3332#15 121 119 JaL731pMT3332#16 ND ND JaL731pMT3332#17 131 120 JaL731pMT3332#18 139 157 JaL731pMT3332#19 126 163 JaL731pMT3332#20 50 ND JaL731pMT3332#21 94 ND JaL731pMT3332#22 118 166 JaL731pMT3332#23 72 56 JaL731pMT3332#24 123 95 JaL731 100 100

[0228]The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Example 4

Construction of a ZY082808 Deleted A. Oryzae Strain MT3420 and Expression of a Variant Humicula Lanuginose Lipase

[0229]First a ZY082808 deleted A. oryzae strain, MT3420, was constructed. The sequences flanking the ZY082808 ORF were PCR amplified with primers based on the published A. oryzae genomic sequence (Machida, M. et al. Nature 438 (2005)1157-1161) using genomic DNA from A. oryzae strain BECh2 as template. The 5' flank was amplified using primers 905 and 904 (SEQ ID NO: 31 and 30) to give a PCR fragment of 1162 bp, whereas the 3'flank was amplified by primers 907 and 906 (SEQ ID NO: 33 and 32) to give an 1150 by fragment. Primers were designed to allow subsequent SOE PCR using primers 905 and 906 to join the fragments placing a Hind3 site between the two flanks. The SOE assembled fragment (2.2 kb) was cloned into the pCR4 TOPO blunt (InVitrogen) to give pMT3369 (SEQ ID NO: 26). The repeat flanked pyrG selection marker was inserted as a Hind3-Hind3 fragment (as described in example 1) into the unique Hind3 site of pMT3369 to give plasmid pMT3376 (SEQ ID NO: 27). Finally the deletion cassette of pMT3376 containing the two flanks of ZY082808 on either side of the pyrG selection marker was transferred as a Not1-Spe1 fragment into the Not1-Xba1 sites of the tk counterselectable plasmid pJaL504 to give the deletion plasmid pMT3378 (SEQ ID NO: 28).Note that pMT3378 contains a unique Not1 site immediately downstream of the 3'flank. This can be used to linearize the plasmid prior to transformation into A. oryzae.

[0230]The linearized plasmid pMT3378 was transformed into A. oryzae JaL355, as described in WO2007045248, by selection for pyrG and counter selection for the tk gene on FDU plates as previously described. 34 transformant colonies were twice reisolated and finally grown up on liquid medium (YPD). Chromosomal DNA was prepared and used in the first place for PCR analysis of the ZY082808 locus. Primers 995 and 996 (SEQ ID NO: 34 and 35) were used for the amplification. The amplified band is expected to have a size of 3.32 kb in the wt strain, while a band of 5.89 kb is expected when the pMT3378 derived deletion cassette has been inserted. Seven of the 34 selected colonies showed the band expected for the deletion strain.

[0231]Four potential deletion strains were chosen for Southern verification of the deletion. DNA from a non-deleted control A. oryzae strain was run in parallel. The genomic DNA was digested with either BamH1 or with BgI2 and probed with a PCR fragment generated from primers 999 and 1000 (SEQ ID NO: 36 and 37) (A. oryzae BECh2 template). In the BamH1 digested samples of the control strain the probe hybridized to the expected size band of 3.9 kb while three out of the four potentially ZY082808 deleted strains showed the predicted bands of 2.0 kb and 1.35 kb. In the BgI2 digest the hybridizing bands were 4.7 kb for the control and 6.4 kb plus 0.9 kb for the same three potentially deleted strains. The fourth deletion candidate strain showed a more complex pattern presumably due to a rearrangement during the integration event. This fourth strain was discarded. Two of the strains showing a clean ZY082808 disruption in the Southern were frozen to the strain collection as MT3420 and 3421.

[0232]MT3420 was transformed with pMT3431 (SEQ ID NO: 29) selecting for the amdS marker. pMT3431 is an expression plasmid for the variant Humicola lanuginose lipase described under construction of pJaL958, example 1. The main difference between pJaL958 and pMT3431 is the marker for selection which in pMT3431 is the A. nidulans amdS gene.

[0233]Transformants were obtained and twice reisolated through sporulation. The reisolated transformants were grown and tested for their expression level of the lipase variant.

[0234]Transformants of the parent strain A. oryzae Bech2 were obtained and tested for parallel comparisons.

Shake Flask Evaluation

[0235]Two times ten lipase transformants made in respectively MT3420 and BECh2 were tested in shake flask experiments by measurement of lipase activity (LU/ml). The same set up as described in example 2 was applied except that both 1/5MDU2B and 1/5GDU2B shake flask medium were used for expression. 1/5GDU2B is composed as 1/5MDU2B apart from maltose has been substituted with glucose.

[0236]In the first experiment the series of transformants in MT3420 had an approx. 20% improved expression in maltose fermentations judged by average and median compared to the series in BECh2 as shown in Table 5. No difference between the hosts was observed in glucose fermentations, meaning that the maltose/glucose expression ratio was increased for transformants in MT3420.

[0237]Also in the second shake flask experiment a somewhat increased maltose/glucose expression ratio was observed for the series of transformants in MT3420 compared to the series in BECh2. The experiments have been hampered of variation originating from the none-robust lipase analysis and the nature of the transformants.

[0238]In conclusion, there is indication of improved performance for the host MT3420.

TABLE-US-00008 TABLE 5 Expression of 10 transformants in MT3420 and 10 transformants in BECh2 ranked after normalization proportionate to the highest expressing transformant in 1/5MDU2B in two independent experiments. 1/5MDU2B 1/5 GDU2B MT3420 BECH2 MT3420 BECH2 Experiment 1 1 100 68 150 141 2 92 60 128 141 3 69 56 122 130 4 61 53 109 121 5 59 52 96 91 6 51 52 95 87 7 45 44 89 83 8 45 34 71 65 9 40 33 41 25 10 33 26 38 8 Experiment 2 1 85 100 86 128 2 77 98 85 109 3 75 95 82 102 4 73 82 80 72 5 71 75 75 65 6 58 39 62 65 7 57 38 60 58 8 52 34 53 35 9 51 21 44 19 10 40 7 23 5

Sequence CWU 1

371240DNAAspergillus oryzae 1atgagactct ccctcaacct tctactgatc gtcggatctg ccgcagtcgc ccgggctgct 60ctggtcccgg tcccgggggc tagcgaagaa ctttgcggac gtcttggagt catgtattac 120gatcctgata atctccccga gggtgtggag gtgcatgaga tacggaagtg tgccggacat 180cccatgggtc gcgagaacta ttggggcttg ggtgattatc tgccgaggtg gtttccttag 240279PRTAspergillu oryzae 2Met Arg Leu Ser Leu Asn Leu Leu Leu Ile Val Gly Ser Ala Ala Val1 5 10 15Ala Arg Ala Ala Leu Val Pro Val Pro Gly Ala Ser Glu Glu Leu Cys 20 25 30Gly Arg Leu Gly Val Met Tyr Tyr Asp Pro Asp Asn Leu Pro Glu Gly 35 40 45Val Glu Val His Glu Ile Arg Lys Cys Ala Gly His Pro Met Gly Arg 50 55 60Glu Asn Tyr Trp Gly Leu Gly Asp Tyr Leu Pro Arg Trp Phe Pro65 70 75360DNAArtificial SequencePCR primer 3gacgaattct ctagaagatc tctcgaggag ctcaagcttc tgtacagtga ccggtgactc 60427DNAArtificial SequencePCR primer 4gacgaattcc gatgaatgtg tgtcctg 27530DNAArtificial SequencePCR primer 5tcttggtacc ctcagcaaag ccatcattgc 30632DNAArtificial SequencePCR primer 6tctctctaga ctccctctgg tttaatactc cg 32727DNAArtificial SequencePCR primer 7catttggatc caccatgaga ctctccc 27829DNAArtificial SequencePCR primer 8ttctctcgag ctaaggaaac cacctcggc 29935DNAArtificial SequencePCR primer 9tcttggatcc gtcgaccttg gggaagtcat caccg 351033DNAArtificial SequencePCR primer 10gtcattaatc attgaattct cgtcgcgact ctc 331145DNAArtificial SequencePCR primer 11gtcgcgacga gaattcaatg attaatgacc ttttcctaaa tatac 451240DNAArtificial SequencePCR primer 12tcttctcgag cctaggtatc ccaaagtcaa gtacgtcagg 401337DNAArtificial SequencePCR primer 13tcttctcgag cctaggctct ccctcaacct tctactg 37146022DNAArtificial Sequencecomplete DNA sequence of plasmid pJaL504 14gaatacacgg aattcctcga gtaccattta attctatttg tgtttgatcg agacctaata 60cagcccctac aacgaccatc aaagtcgtat agctaccagt gaggaagtgg actcaaatcg 120acttcagcaa catctcctgg ataaacttta agcctaaact atacagaata agataggtgg 180agagcttata ccgagctccc aaatctgtcc agatcatggt tgaccggtgc ctggatcttc 240ctatagaatc atccttattc gttgacctag ctgattctgg agtgacccag agggtcatga 300cttgagccta aaatccgccg cctccaccat ttgtagaaaa atgtgacgaa ctcgtgagct 360ctgtacagtg accggtgact ctttctggca tgcggagaga cggacggacg cagagagaag 420ggctgagtaa taagccactg gccagacagc tctggcggct ctgaggtgca gtggatgatt 480attaatccgg gaccggccgc ccctccgccc cgaagtggaa aggctggtgt gcccctcgtt 540gaccaagaat ctattgcatc atcggagaat atggagcttc atcgaatcac cggcagtaag 600cgaaggagaa tgtgaagcca ggggtgtata gccgtcggcg aaatagcatg ccattaacct 660aggtacagaa gtccaattgc ttccgatctg gtaaaagatt cacgagatag taccttctcc 720gaagtaggta gagcgagtac ccggcgcgta agctccctaa ttggcccatc cggcatctgt 780agggcgtcca aatatcgtgc ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc 840tcaggagctg gccagcggcg cagaccggga acacaagctg gcagtcgacc catccggtgc 900tctgcactcg acctgctgag gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc 960gcccggtgtg tcggcggggt tgacaaggtc gttgcgtcag tccaacattt gttgccatat 1020tttcctgctc tccccaccag ctgtagatct tggtggcgtg aaactcccgc acctcttcgg 1080ccagcgcctt gtagaagcgc gtatggcttc gtaccccggc catcaacacg cgtctgcgtt 1140cgaccaggct gcgcgttctc gcggccatag caaccgacgt acggcgttgc gccctcgccg 1200gcagcaagaa gccacggaag tccgcccgga gcagaaaatg cccacgctac tgcgggttta 1260tatagacggt ccccacggga tggggaaaac caccaccacg caactgctgg tggccctggg 1320ttcgcgcgac gatatcgtct acgtacccga gccgatgact tactggcggg tgctgggggc 1380ttccgagaca atcgcgaaca tctacaccac acaacaccgc ctcgaccagg gtgagatatc 1440ggccggggac gcggcggtgg taatgacaag cgcccagata acaatgggca tgccttatgc 1500cgtgaccgac gccgttctgg ctcctcatat cgggggggag gctgggagct cacatgcccc 1560gcccccggcc ctcaccctca tcttcgaccg ccatcccatc gccgccctcc tgtgctaccc 1620ggccgcgcgg taccttatgg gcagcatgac cccccaggcc gtgctggcgt tcgtggccct 1680catcccgccg accttgcccg gcaccaacat cgtgcttggg gcccttccgg aggacagaca 1740catcgaccgc ctggccaaac gccagcgccc cggcgagcgg ctggacctgg ctatgctggc 1800tgcgattcgc cgcgtttacg ggctacttgc caatacggtg cggtatctgc agtgcggcgg 1860gtcgtggcgg gaggactggg gacagctttc ggggacggcc gtgccgcccc agggtgccga 1920gccccagagc aacgcgggcc cacgacccca tatcggggac acgttattta ccctgtttcg 1980gggccccgag ttgctggccc ccaacggcga cctgtataac gtgtttgcct gggccttgga 2040cgtcttggcc aaacgcctcc gttccatgca cgtctttatc ctggattacg accaatcgcc 2100cgccggctgc cgggacgccc tgctgcaact tacctccggg atggtccaga cccacgtcac 2160cacccccggc tccataccga cgatatgcga cctggcgcgc acgtttgccc gggagatggg 2220ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atccggatcg 2280atccacttaa cgttactgaa atcatcaaac agcttgacga atctggatat aagatcgttg 2340gtgtcgatgt cagctccgga gttgagacaa atggtgttca ggatctcgat aagatacgtt 2400catttgtcca agcagcaaag agtgccttct agtgatttaa tagctccatg tcaacaagaa 2460taaaacgcgt tttcgggttt acctcttcca gatacagctc atctgcaatg cattaatgca 2520ttgactgcaa cctagtaacg ccttncaggc tccggcgaag agaagaatag cttagcagag 2580ctattttcat tttcgggaga cgagatcaag cagatcaacg gtcgtcaaga gacctacgag 2640actgaggaat ccgctcttgg ctccacgcga ctatatattt gtctctaatt gtactttgac 2700atgctcctct tctttactct gatagcttga ctatgaaaat tccgtcacca gcncctgggt 2760tcgcaaagat aattgcatgt ttcttccttg aactctcaag cctacaggac acacattcat 2820cgtaggtata aacctcgaaa tcanttccta ctaagatggt atacaatagt aaccatgcat 2880ggttgcctag tgaatgctcc gtaacaccca atacgccggc cgaaactttt ttacaactct 2940cctatgagtc gtttacccag aatgcacagg tacacttgtt tagaggtaat ccttctttct 3000agaagtcctc gtgtactgtg taagcgccca ctccacatct ccactcgacc tgcaggcatg 3060caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 3120ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 3180gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 3240gccagagcgg ccgctctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 3300attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 3360cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 3420gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 3480ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 3540agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 3600tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 3660ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 3720gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 3780ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 3840gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 3900aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 3960aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 4020ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 4080gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 4140gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 4200tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 4260ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 4320ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 4380atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 4440ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 4500tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 4560attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 4620tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 4680ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 4740gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 4800gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 4860gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 4920aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 4980taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 5040tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 5100tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 5160atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 5220tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 5280aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac 5340ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc 5400agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat 5460gcggcatcag agcagattgt actgagagtg caccatatcg acgctctccc ttatgcgact 5520cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga 5580atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac 5640ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga 5700tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc 5760gtccggcgta gaggatctgg ctagcgatga ccctgctgat tggttcgctg accatttccg 5820gggtgcggaa cggcgttacc agaaactcag aaggttcgtc caaccaaacc gactctgacg 5880gcagtttacg agagagatga tagggtctgc ttcagtaagc cagatgctac acaattaggc 5940ttgtacatat tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac 6000atacgattta ggtgacacta ta 6022156471DNAArtificial Sequencecomplete DNA sequence of plasmid pJaL575 15agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca 240gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc ccttgacgaa 300ttctctagaa gatctctcga ggagctcaag cttgagctct gtacagtgac cggtgactct 360ttctggcatg cggagagacg gacggacgca gagagaaggg ctgagtaata acggccactg 420cgccagacag ctctggcggc tctgaggtgc agtggatgat tattaatccg ggaccggccg 480cccctccgcc ccgaagtgga aaggctggtg tgcccctcgt tgaccaagaa tctattgcat 540catcggagaa tatggagctt catcgaatca ccggcagtaa gcgaaggaga atgtgaagcc 600aggggtgtat agccgtcggc gaaatagcat gccattaacc taggtacaga agtccaattg 660cttccgatct ggtaaaagat tcacgagata gtaccttctc cgaagtaggt agagcgagta 720cccggcgcgt aagctcccta attggcccat ccggcatctg tagggcgtcc aaatatcgtg 780cctctcctgc tttgcccggt gtatgaaacc ggaaaggccg ctcaggagct ggccagcggc 840gcagaccggg aacacaagct ggcagtcgac ccatccggtg ctctgcactc gacctgctga 900ggtccctcag tccctggtag gcagctttgc cccgtctgtc cgcccggtgt gtcggcgggg 960ttgacaaggt cgttgcgtca gtccaacatt tgttgccata ttttcctgct ctccccacca 1020gctgtagatc gatcttggtg gcgtgaaact cccgcacctc ttcggccagc gccttgtaga 1080agcgcgtatg gcttcgtacc ccggccatca acacgcgtct gcgttcgacc aggctgcgcg 1140ttctcgcggc catagcaacc gacgtacggc gttgcgccct cgccggcagc aagaagccac 1200ggaagtccgc ccggagcaga aaatgcccac gctactgcgg gtttatatag acggtcccca 1260cgggatgggg aaaaccacca ccacgcaact gctggtggcc ctgggttcgc gcgacgatat 1320cgtctacgta cccgagccga tgacttactg gcgggtgctg ggggcttccg agacaatcgc 1380gaacatctac accacacaac accgcctcga ccagggtgag atatcggccg gggacgcggc 1440ggtggtaatg acaagcgccc agataacaat gggcatgcct tatgccgtga ccgacgccgt 1500tctggctcct catatcgggg gggaggctgg gagctcacat gccccgcccc cggccctcac 1560cctcatcttc gaccgccatc ccatcgccgc cctcctgtgc tacccggccg cgcggtacct 1620tatgggcagc atgacccccc aggccgtgct ggcgttcgtg gccctcatcc cgccgacctt 1680gcccggcacc aacatcgtgc ttggggccct tccggaggac agacacatcg accgcctggc 1740caaacgccag cgccccggcg agcggctgga cctggctatg ctggctgcga ttcgccgcgt 1800ttacgggcta cttgccaata cggtgcggta tctgcagtgc ggcgggtcgt ggcgggagga 1860ctggggacag ctttcgggga cggccgtgcc gccccagggt gccgagcccc agagcaacgc 1920gggcccacga ccccatatcg gggacacgtt atttaccctg tttcggggcc ccgagttgct 1980ggcccccaac ggcgacctgt ataacgtgtt tgcctgggcc ttggacgtct tggccaaacg 2040cctccgttcc atgcacgtct ttatcctgga ttacgaccaa tcgcccgccg gctgccggga 2100cgccctgctg caacttacct ccgggatggt ccagacccac gtcaccaccc ccggctccat 2160accgacgata tgcgacctgg cgcgcacgtt tgcccgggag atgggggagg ctaactgaaa 2220cacggaagga gacaataccg gaaggaaccc gcgctatccg gatcgatcca cttaacgtta 2280ctgaaatcat caaacagctt gacgaatctg gatataagat cgttggtgtc gatgtcagct 2340ccggagttga gacaaatggt gttcaggatc tcgataagat acgttcattt gtccaagcag 2400caaagagtgc cttctagtga tttaatagct ccatgtcaac aagaataaaa cgcgtttcgg 2460gtttacctct tccagataca gctcatctgc aatgcattaa tgcattggac ctcgcaaccc 2520tagtacgccc ttcaggctcc ggcgaagcag aagaatagct tagcagagtc tattttcatt 2580ttcgggagac gagatcaagc agatcaacgg tcgtcaagag acctacgaga ctgaggaatc 2640cgctcttggc tccacgcgac tatatatttg tctctaattg tactttgaca tgctcctctt 2700ctttactctg atagcttgac tatgaaaatt ccgtcaccag cncctgggtt cgcaaagata 2760attgcactgt ttcttccttg aactctcaag cctacaggag aattcgtcaa gggcgaattc 2820gcggccgcta aattcaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt 2880ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 2940ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 3000ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga gagccgttat 3060cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg gatggtgatc 3120cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg 3180catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc 3240gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt 3300aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag gatcttcacc 3360tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc 3420agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc 3480agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa 3540ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct 3600ttctcgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga gacaggatga 3660ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg 3720gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg 3780ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc 3840ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct 3900tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa 3960gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg 4020gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa 4080gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat 4140gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg 4200agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc 4260atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac 4320cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg 4380gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc 4440tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct gatgcggtat 4500tttctcctta cgcatctgtg cggtatttca caccgcatac aggtggcact tttcggggaa 4560atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 4620tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 4680aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 4740acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 4800acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 4860ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 4920ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 4980caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 5040ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 5100aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 5160aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 5220tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 5280aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 5340cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 5400ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 5460gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 5520agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 5580atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 5640cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 5700cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 5760cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 5820tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 5880tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 5940ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 6000aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 6060cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 6120ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 6180agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 6240ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 6300acgcggcctt tttacggttc ctgggctttt gctggccttt tgctcacatg ttctttcctg 6360cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 6420gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g 6471166169DNAArtificial Sequencecomplete DNA sequence of plasmid pJaL731 16tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccaggccaag ctttcgcgag ctcgagatct 420gtataaagaa ggtatgagtt ataactgctc cactcctatc cttatcaggt tgcttgcacc 480ggctgtcatg cttgtcccct tgagccgtta cattctcaca ctctgaaagg gtttttaaca 540tcgccggagc tcttctatgt

tcgaaatcat ggcccagtcc ctcatatcaa ggatgaagat 600atccctcact gggaaattag catcgaaggg ttagtatagt gctaggttct ctgccaaaca 660tccgttaacc aaagtataga ctggtagaga agcctttggt actaaacttc cgacaagtgt 720tgcagcagta cgaccaaata acagcgccta tcaccctcgt atgtgctggc aatcgacgca 780aagagcaaaa cattgtacgt aaaacgaaag gtttttcctg gggatcggcg ggactatcga 840ctgccctctt cactggccca ttgctggcgg atattctccg cagtgcgaaa cccctgcgta 900aagcgaaata cgtctgtatg gaaggagcgg ataagctggt atgctgtacc tctatcttat 960gatgataatt gctaagttcg ccgcagccca atggtcacta cggcacatct attaaattga 1020actgggccct ggaccccaac agggggatca tgcttgcaca taaaatgaac ggggagtctc 1080ttcgcccaga tcatggtcgt ccgctgaggg ccgtcgtgcc cggtcaaata ggaggacgaa 1140gtgttaagtg gctgaagagg ctgatcttga ccgatgcacc aagcgacaac tggtacagct 1200tatcagctgc atatctctat atctccttat catcctgaca tccacttact aaagtgactg 1260cagtgggtcc tgctaccttt ccccgtcaag gagcttatcg atagcagtac tagcgcgtgc 1320gctgtaggtc aattgcgact tggaagacat gttggcgatg gaggggtagc gcggggttct 1380gcaaatattg tataaatgag cacttagtgg ttgaaactgg cttattagta ggttagtact 1440tcgagttttc agtaattaga caaaataatc aggatgtcca actactaact cttgatatat 1500ggaatgaaat gtagatacaa actgcacgac aattgccgcg aaaaattaaa ttgaatctat 1560ggaggggact gtcatgcact agccacacgt ttctccgcct gtggggtgag ccacatgcct 1620cattttgacc aaacacatcg atgcagtcac atgtagataa gattagggcc tatccttagg 1680gtacattagt gataccccac tctaagaaaa tagaccaatc tccagctgca ccttcagaca 1740ctccggtaca aattctcgtc tatgttggag attgttgtga ctttgaaaca tgacccttga 1800ccctgatttt gaatttgtcc atatatcgag gcaggtgtct tattcgtacg gagagggtat 1860ctgtcgtaga cacatagtag tagtcatttc gagtgctgaa tttataaatc gcatcatact 1920tgcgacatac tgccataaaa ggagtacgta tccaccacta cttattgcgc accaacacgc 1980ttcaggtatg catcccatcc ctccttctgg tactgcttcg ccgcctccac gggatcagga 2040gcagcataaa ttccacggcc agcaataata aagtcggcac cgcgtccaac agccgactca 2100ggagtttggt actgctgtcc cagcttgtca cccttcgagg agaggttgac acctgtcgtg 2160aagacgacaa aatcttcctc ctccgaaggc gagctaactt cagactgaac ctcgccaagg 2220tgacgtgtcg agacgaatcc catcacaaac ttcttatact tccgagcata gtcaacagaa 2280gaagtagtat attgaccggt agccaaagat cccttggagg tcatctccgc aaggatcaaa 2340aggcccctct cggagccgta ggggaagtcc tcggccgaag cagtctgggc cagagcctcg 2400acgataccct caccgggcag aatactgcag ttgatgatgt gggcccactc agagatacgc 2460agagtgccgc catggtactg cttttggact gtgtttccga tatcgatgaa cttgcgatct 2520tcgaagatga ggaaattgtg cttctctgca agggccttca gaccggtgat ggtttcttcg 2580ctgaaatcgg agaggatatc gatgtgagtt ttgatcacgg caatgtacgg accgagtcct 2640gttatataat ccaccattaa ccattactag atcacatgta agtggcatcc ccggtgcgca 2700tacggtcagc caaatccagc agctctttgg tggttgtcac gtcggcggaa acggtgacat 2760tggttttctt ggcctcggca acctcgaaga gcttctttac gagcgcattg gggtgcttgc 2820tagcgcgtgc gctgtaggtc aattgcgact tggaagacat gttggcgatg gaggggtagc 2880gcggggttct gcaaatattg tataaatgag cacttagtgg ttgaaactgg cttattagta 2940ggttagtact tcgagttttc agtaattaga caaaataatc aggatgtcca actactaact 3000cttgatatat ggaatgaaat gtagatacaa actgcacgac aattgccgcg aaaaattaaa 3060ttgaatctat ggaggggact gtcatgcact agccacacgt ttctccgcct gtggggtgag 3120ccacatgcct cattttgacc aaacacatcg atgcagtcac atgtagataa gattagggcc 3180tatccttagg gtaccgcgct aggaggtggt cgaatacaca tgtaggcgct tctctggatg 3240caaaagtcgt gccggacctg ccgaaagact ttgaagatgc gttcacgcca tctaagttgc 3300gtagataatt cacaaaaagg gatgtttgtt tccggaatgt agcaaagagc tgataggcaa 3360tagcctcact ttcgtggcgc acgccgctcg ttccatccat cctcgacaat ggagcaaatg 3420tcaaaatcgt accgaaaata ctttccagca gcttcgctgc atcagcatgt cttttgctga 3480gaaagagcgc aaaaagcatt tgatcgagaa tatcttcatg ataatctcta agtctaggga 3540cagaatgtgc tgcttctatc gtgccatcaa tatcaccgcg gtcgaggcag cgttcaatct 3600tagccaggct atcttggaac cgctgccaag tcgagccaat gccgacatga aagcaataat 3660cactcaatga gagcacgaaa tgctggcagt caatgcgaaa tttctggtac acgtttcgag 3720ggtgcccaga tagggagtct ctccccgtag aatcacgaat gagacctttg acgaccgaaa 3780ccattcgaag gagtcgaagc agatgcttga aaagacgatc atacttgtta agcgatcgcg 3840acgtaatgat agcttccagg acgtctgatg gtttgtattg aagacgtagg aaatccaaag 3900cttgcatgcc tgcaggtcga cctgcagcca agcttggcgt aatcatggtc atagctgttt 3960cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 4020tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 4080cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 4140gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 4200tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 4260acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 4320aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 4380cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 4440gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 4500tacctgtccg cctttttccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 4560tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 4620cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 4680gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 4740ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 4800ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 4860ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 4920agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 4980aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 5040atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 5100tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5160tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 5220tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 5280gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 5340tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 5400ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 5460gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 5520aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 5580ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 5640tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 5700ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 5760aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 5820ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 5880ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 5940agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 6000tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 6060ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt 6120atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc 6169179971DNAArtificial Sequencecomplete DNA sequence of plasmid pJaL736 17agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca 240gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc ccttgacgaa 300ttctctagaa gatctctcga ggagctcaag ctttcgcgag ctcgagatct gtataaagaa 360ggtatgagtt ataactgctc cactcctatc cttatcaggt tgcttgcacc ggctgtcatg 420cttgtcccct tgagccgtta cattctcaca ctctgaaagg gtttttaaca tcgccggagc 480tcttctatgt tcgaaatcat ggcccagtcc ctcatatcaa ggatgaagat atccctcact 540gggaaattag catcgaaggg ttagtatagt gctaggttct ctgccaaaca tccgttaacc 600aaagtataga ctggtagaga agcctttggt actaaacttc cgacaagtgt tgcagcagta 660cgaccaaata acagcgccta tcaccctcgt atgtgctggc aatcgacgca aagagcaaaa 720cattgtacgt aaaacgaaag gtttttcctg gggatcggcg ggactatcga ctgccctctt 780cactggccca ttgctggcgg atattctccg cagtgcgaaa cccctgcgta aagcgaaata 840cgtctgtatg gaaggagcgg ataagctggt atgctgtacc tctatcttat gatgataatt 900gctaagttcg ccgcagccca atggtcacta cggcacatct attaaattga actgggccct 960ggaccccaac agggggatca tgcttgcaca taaaatgaac ggggagtctc ttcgcccaga 1020tcatggtcgt ccgctgaggg ccgtcgtgcc cggtcaaata ggaggacgaa gtgttaagtg 1080gctgaagagg ctgatcttga ccgatgcacc aagcgacaac tggtacagct tatcagctgc 1140atatctctat atctccttat catcctgaca tccacttact aaagtgactg cagtgggtcc 1200tgctaccttt ccccgtcaag gagcttatcg atagcagtac tagcgcgtgc gctgtaggtc 1260aattgcgact tggaagacat gttggcgatg gaggggtagc gcggggttct gcaaatattg 1320tataaatgag cacttagtgg ttgaaactgg cttattagta ggttagtact tcgagttttc 1380agtaattaga caaaataatc aggatgtcca actactaact cttgatatat ggaatgaaat 1440gtagatacaa actgcacgac aattgccgcg aaaaattaaa ttgaatctat ggaggggact 1500gtcatgcact agccacacgt ttctccgcct gtggggtgag ccacatgcct cattttgacc 1560aaacacatcg atgcagtcac atgtagataa gattagggcc tatccttagg gtacattagt 1620gataccccac tctaagaaaa tagaccaatc tccagctgca ccttcagaca ctccggtaca 1680aattctcgtc tatgttggag attgttgtga ctttgaaaca tgacccttga ccctgatttt 1740gaatttgtcc atatatcgag gcaggtgtct tattcgtacg gagagggtat ctgtcgtaga 1800cacatagtag tagtcatttc gagtgctgaa tttataaatc gcatcatact tgcgacatac 1860tgccataaaa ggagtacgta tccaccacta cttattgcgc accaacacgc ttcaggtatg 1920catcccatcc ctccttctgg tactgcttcg ccgcctccac gggatcagga gcagcataaa 1980ttccacggcc agcaataata aagtcggcac cgcgtccaac agccgactca ggagtttggt 2040actgctgtcc cagcttgtca cccttcgagg agaggttgac acctgtcgtg aagacgacaa 2100aatcttcctc ctccgaaggc gagctaactt cagactgaac ctcgccaagg tgacgtgtcg 2160agacgaatcc catcacaaac ttcttatact tccgagcata gtcaacagaa gaagtagtat 2220attgaccggt agccaaagat cccttggagg tcatctccgc aaggatcaaa aggcccctct 2280cggagccgta ggggaagtcc tcggccgaag cagtctgggc cagagcctcg acgataccct 2340caccgggcag aatactgcag ttgatgatgt gggcccactc agagatacgc agagtgccgc 2400catggtactg cttttggact gtgtttccga tatcgatgaa cttgcgatct tcgaagatga 2460ggaaattgtg cttctctgca agggccttca gaccggtgat ggtttcttcg ctgaaatcgg 2520agaggatatc gatgtgagtt ttgatcacgg caatgtacgg accgagtcct gttatataat 2580ccaccattaa ccattactag atcacatgta agtggcatcc ccggtgcgca tacggtcagc 2640caaatccagc agctctttgg tggttgtcac gtcggcggaa acggtgacat tggttttctt 2700ggcctcggca acctcgaaga gcttctttac gagcgcattg gggtgcttgc tagcgcgtgc 2760gctgtaggtc aattgcgact tggaagacat gttggcgatg gaggggtagc gcggggttct 2820gcaaatattg tataaatgag cacttagtgg ttgaaactgg cttattagta ggttagtact 2880tcgagttttc agtaattaga caaaataatc aggatgtcca actactaact cttgatatat 2940ggaatgaaat gtagatacaa actgcacgac aattgccgcg aaaaattaaa ttgaatctat 3000ggaggggact gtcatgcact agccacacgt ttctccgcct gtggggtgag ccacatgcct 3060cattttgacc aaacacatcg atgcagtcac atgtagataa gattagggcc tatccttagg 3120gtaccgcgct aggaggtggt cgaatacaca tgtaggcgct tctctggatg caaaagtcgt 3180gccggacctg ccgaaagact ttgaagatgc gttcacgcca tctaagttgc gtagataatt 3240cacaaaaagg gatgtttgtt tccggaatgt agcaaagagc tgataggcaa tagcctcact 3300ttcgtggcgc acgccgctcg ttccatccat cctcgacaat ggagcaaatg tcaaaatcgt 3360accgaaaata ctttccagca gcttcgctgc atcagcatgt cttttgctga gaaagagcgc 3420aaaaagcatt tgatcgagaa tatcttcatg ataatctcta agtctaggga cagaatgtgc 3480tgcttctatc gtgccatcaa tatcaccgcg gtcgaggcag cgttcaatct tagccaggct 3540atcttggaac cgctgccaag tcgagccaat gccgacatga aagcaataat cactcaatga 3600gagcacgaaa tgctggcagt caatgcgaaa tttctggtac acgtttcgag ggtgcccaga 3660tagggagtct ctccccgtag aatcacgaat gagacctttg acgaccgaaa ccattcgaag 3720gagtcgaagc agatgcttga aaagacgatc atacttgtta agcgatcgcg acgtaatgat 3780agcttccagg acgtctgatg gtttgtattg aagacgtagg aaatccaaag cttgagctct 3840gtacagtgac cggtgactct ttctggcatg cggagagacg gacggacgca gagagaaggg 3900ctgagtaata agcgccactg cgccagacag ctctggcggc tctgaggtgc agtggatgat 3960tattaatccg ggaccggccg cccctccgcc ccgaagtgga aaggctggtg tgcccctcgt 4020tgaccaagaa tctattgcat catcggagaa tatggagctt catcgaatca ccggcagtaa 4080gcgaaggaga atgtgaagcc aggggtgtat agccgtcggc gaaatagcat gccattaacc 4140taggtacaga agtccaattg cttccgatct ggtaaaagat tcacgagata gtaccttctc 4200cgaagtaggt agagcgagta cccggcgcgt aagctcccta attggcccat ccggcatctg 4260tagggcgtcc aaatatcgtg cctctcctgc tttgcccggt gtatgaaacc ggaaaggccg 4320ctcaggagct ggccagcggc gcagaccggg aacacaagct ggcagtcgac ccatccggtg 4380ctctgcactc gacctgctga ggtccctcag tccctggtag gcagctttgc cccgtctgtc 4440cgcccggtgt gtcggcgggg ttgacaaggt cgttgcgtca gtccaacatt tgttgccata 4500ttttcctgct ctccccacca gctgtagatc gatcttggtg gcgtgaaact cccgcacctc 4560ttcggccagc gccttgtaga agcgcgtatg gcttcgtacc ccggccatca acacgcgtct 4620gcgttcgacc aggctgcgcg ttctcgcggc catagcaacc gacgtacggc gttgcgccct 4680cgccggcagc aagaagccac ggaagtccgc ccggagcaga aaatgcccac gctactgcgg 4740gtttatatag acggtcccca cgggatgggg aaaaccacca ccacgcaact gctggtggcc 4800ctgggttcgc gcgacgatat cgtctacgta cccgagccga tgacttactg gcgggtgctg 4860ggggcttccg agacaatcgc gaacatctac accacacaac accgcctcga ccagggtgag 4920atatcggccg gggacgcggc ggtggtaatg acaagcgccc agataacaat gggcatgcct 4980tatgccgtga ccgacgccgt tctggctcct catatcgggg gggaggctgg gagctcacat 5040gccccgcccc cggccctcac cctcatcttc gaccgccatc ccatcgccgc cctcctgtgc 5100tacccggccg cgcggtacct tatgggcagc atgacccccc aggccgtgct ggcgttcgtg 5160gccctcatcc cgccgacctt gcccggcacc aacatcgtgc ttggggccct tccggaggac 5220agacacatcg accgcctggc caaacgccag cgccccggcg agcggctgga cctggctatg 5280ctggctgcga ttcgccgcgt ttacgggcta cttgccaata cggtgcggta tctgcagtgc 5340ggcgggtcgt ggcgggagga ctggggacag ctttcgggga cggccgtgcc gccccagggt 5400gccgagcccc agagcaacgc gggcccacga ccccatatcg gggacacgtt atttaccctg 5460tttcggggcc ccgagttgct ggcccccaac ggcgacctgt ataacgtgtt tgcctgggcc 5520ttggacgtct tggccaaacg cctccgttcc atgcacgtct ttatcctgga ttacgaccaa 5580tcgcccgccg gctgccggga cgccctgctg caacttacct ccgggatggt ccagacccac 5640gtcaccaccc ccggctccat accgacgata tgcgacctgg cgcgcacgtt tgcccgggag 5700atgggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatccg 5760gatcgatcca cttaacgtta ctgaaatcat caaacagctt gacgaatctg gatataagat 5820cgttggtgtc gatgtcagct ccggagttga gacaaatggt gttcaggatc tcgataagat 5880acgttcattt gtccaagcag caaagagtgc cttctagtga tttaatagct ccatgtcaac 5940aagaataaaa cgcgtttcgg gtttacctct tccagataca gctcatctgc aatgcattaa 6000tgcattggac ctcgcaaccc tagtacgccc ttcaggctcc ggcgaagcag aagaatagct 6060tagcagagtc tattttcatt ttcgggagac gagatcaagc agatcaacgg tcgtcaagag 6120acctacgaga ctgaggaatc cgctcttggc tccacgcgac tatatatttg tctctaattg 6180tactttgaca tgctcctctt ctttactctg atagcttgac tatgaaaatt ccgtcaccag 6240cncctgggtt cgcaaagata attgcactgt ttcttccttg aactctcaag cctacaggag 6300aattcgtcaa gggcgaattc gcggccgcta aattcaattc gccctatagt gagtcgtatt 6360acaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 6420ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca 6480ccgatcgccc ttcccaacag ttgcgcagcc tatacgtacg gcagtttaag gtttacacct 6540ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc 6600cggggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc 6660gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata 6720tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa 6780atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca ggcatgagat 6840tatcaaaaag gatcttcacc tagatccttt tcacgtagaa agccagtccg cagaaacggt 6900gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa 6960agagaaagca ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat 7020ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct 7080gcaaagtaaa ctggatggct ttctcgccgc caaggatctg atggcgcagg ggatcaagct 7140ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag 7200gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg 7260gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca 7320agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc 7380tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg 7440actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg 7500ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta 7560cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag 7620ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac 7680tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg 7740atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg 7800gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg 7860aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg 7920attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaatt attaacgctt 7980acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac 8040aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 8100ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 8160aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 8220ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 8280gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 8340ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 8400ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 8460gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 8520aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 8580gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 8640aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 8700caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 8760tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 8820acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 8880gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 8940agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 9000gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 9060ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 9120taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 9180agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 9240aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 9300ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 9360gccgtagtta ggccaccact

tcaagaactc tgtagcaccg cctacatacc tcgctctgct 9420aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 9480aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 9540gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 9600aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 9660aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 9720cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 9780cctatggaaa aacgccagca acgcggcctt tttacggttc ctgggctttt gctggccttt 9840tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 9900tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 9960ggaagcggaa g 9971189321DNAArtificial Sequencecomplete DNA sequence of plasmid pJaL958 18agcttggcgg tgatattgat ggcacgatag aagcagcaca ttctgtccct agacttagag 60attatcatga agatattctc gatcaaatgc tttttgcgct ctttctcagc aaaagacatg 120ctgatgcagc gaagctgctg gaaagtattt tcggtacgat tttgacattt gctccattgt 180cgaggatgga tggaacgagc ggcgtgcgcc acgaaagtga ggctattgcc tatcagctct 240ttgctacatt ccggaaacaa acatcccttt ttgtgaatta tctacgcaac ttagatggcg 300tgaacgcatc ttcaaagtct ttcggcaggt ccggcacgac ttttgcatcc agagaagcgc 360ctacatgtgt attcgaccac ctcctagcgc gcttggatat gaggaaatat tactgagagt 420cgaaaacaag ctccaccgca ccagctcttc ttggagtttt atattaaaga atattcccag 480ctcgttgtat tattcttttt ctaccgtgct aatgtatcaa ggactttggt acctattaac 540gttattattc gtgtgctatt cccaaacata accctgtata tgtttcgaac gccgttatga 600cccatgtctt acatactcat taagtcattc ccttggataa tcccaattta gaagaagtga 660aggtctgatt ctttccatcc ttccgccaac agtatcctcc gagccgattc ttccatggct 720ggcggaccac aaatcaggac catactctca tcttctggag ccgcgtactc ctttaggagc 780tcttcggata tgcgtcctcg gcggccagtc catgagtccg gcgctttgga tagggtgtgt 840attatattac accttctgct gtcggttgcc atgaagccgt cgagctcagc ccggcaaagg 900atatcttcct cctgtctgtt tccattgagg actgtacaag aggtgggatc ttgccggtcc 960tgaaccacgg cgcgcaagac ctggaagatc ggtgtgatac cggttcctcc acaaatcatc 1020ttaaacgacc gaacatggcg ttccttccca cttatgacaa ctcgtccatt tccaaggtat 1080tcgaatctgc ctgtcggacc cttgcattcc accacggagc ccaatggcag cctatccagg 1140gccatcgtca tcttgccgcc tgccgaggtg gctgttgcaa agtatacttt aaccagcaag 1200tccacggtcc ctttctggct ggtttcagaa attggggtgt atgagcggat gatggcttcg 1260ttgttggatg atgtgtcgag gactttgatc ataagatgct ggccgactgg taaacccaat 1320gtttgatctt cgtgttccaa tttgaaacta aatattcgtg tatcccagga tatgtctttc 1380ctttctttca atgttgcctt tgtccaagac cgtgattgga ggaacactgg gcgaatttca 1440tcggtggagg atgatgcatc atccttgagt gcttttaaac cttccgggtc catcgttcca 1500atatggtact caggcatcat cgcctttgcc gtctcgctat ctatggatag gtgtcaatag 1560atggtacaat tgcagtgtga tatttttggg actcacgaat agcaaggaat tcctcagaga 1620catccagacc agcagaggag ataatactct gcgctccgcc agggtggcct tcaagaaatg 1680cttgaccatc atacacttct ccattcacga tgaaccatgg cttctcatcg caggaattct 1740ccttgaattc ttcaaaacca atcactcggc ttagcccgtc tttcttcata ttaatgtctt 1800gcacgggctc cggctccgtc ggctcctctc cttcgtgtct ttctccccag ttaccattcg 1860tcaggtcacc cccagccttt ttgacgcgtt ccatccatcc tgtaggcata ctagggtggg 1920tagggtgctc gaatctcaag ttcccgtttt ccttcgtaat tgtaacccgg aaccacgggt 1980tgttcatcat tccgagaacg gaccagtaca tatcgcgagg ctgcacgccc aatgcttcgt 2040ccatggctct tacaaggatg gcatcactgt tctcaagctc tgggatggtg atgcttagag 2100accaaaaaca ccagcagaag caagtttcgc gccagtacat atctactttg cctccaaaaa 2160gctcgccttc aaaatcacga tacttgtctt cggcatattc gatttccgcc aatctccaag 2220ctataagtcc gttagctttg ataagcattc tcacacatcg agcgagcgag ggtgcgtaca 2280tttgcctttg tctagggata tttctaccct ggtaaccctg cggcccccac cggcgtatgc 2340atatcctctg acagtatatg acggccctgc cgacaggaga tttaagacct cattgttttg 2400gggatatgca acggcggagt tggtgtttag gtcataaatc gcataccgct catcgtgcca 2460ccaatttcgg ttatttgatg ccatctcagg cgagaccatt gttctgggtt agggagttag 2520acaaatgatg gaaatataaa ataagtgccc tttagacata cggtaagacg cggttgtcat 2580tgatatggta ccagttgtcg cttggtgcat cggtcaagat cagcctcttc agccacttaa 2640cacttcgtcc tcctatttga ccgggcacga cggccctcag cggacgacca tgatctgggc 2700gaagagactc cccgttcatt ttatgtgcaa gcatgatccc cctgttgggg tccagggccc 2760agttcaattt aatagatgtg ccgtagtgac cattgggctg cggcgaactt agcaattatc 2820atcataagat agaggtacag cataccagct tatccgctcc ttccatacag acgtatttcg 2880ctttacgcag gggtttcgca ctgcggagaa tatccgccag caatgggcca gtgaagaggg 2940cagtcgatag tcccgccgat ccccaggaaa aacctttcgt tttacgtaca atgttttgct 3000ctttgcgtcg attgccagca catacgaggg tgataggcgc tgttatttgg tcgtactgct 3060gcaacacttg tcggaagttt agtaccaaag gcttctctac cagtctatac tttggttaac 3120ggatgtttgg cagagaacct agcactatac taacccttcg atgctaattt cccagtgagg 3180gatatcttca tccttgatat gagggactgg gccatgattt cgaacataga agagctccgg 3240cgatgttaaa aaccctttca gagtgtgaga atgtaacggc tcaaggggac aagcatgaca 3300gccggtgcaa gcaacctgat aaggatagga gtggagcagt tataactcat accttcttta 3360tacagatctc gagctcgcga aagcttatgg tgttttgatc attttaaatt tttatatggc 3420gggtggtggg caactcgctt gcgcgggcaa ctcgcttacc gattacgtta gggctgatat 3480ttacgtaaaa atcgtcaagg gatgcaagac caaaccgtta aatttccgga gtcaacagca 3540tccaagccca agtccttcac ggagaaaccc cagcgtccac atcacgagcg aaggaccacc 3600tctaggcatc ggacgcacca tccaattaga agcagcaaag cgaaacagcc caagaaaaag 3660gtcggcccgt cggccttttc tgcaacgctg atcacgggca gcgatccaac caacaccctc 3720cagagtgact aggggcggaa atttatcggg attaatttcc actcaaccac aaatcacagt 3780cgtccccggt aatttaacgg ctgcagacgg caatttaacg gcttctgcga atcgcttgga 3840ttccccgccc ctggccgtag agcttaaagt atgtcccttg tcgatgcgat gtatgaatta 3900gcttatggtg ttttgatcat tttaaatttt tatatggcgg gtggtgggca actcgcttgc 3960gcgggcaact cgcttaccga ttacgttagg gctgatattt acgtaaaaat cgtcaaggga 4020tgcaagacca aaccgttaaa tttccggagt caacagcatc caagcccaag tccttcacgg 4080agaaacccca gcgtccacat cacgagcgaa ggaccacctc taggcatcgg acgcaccatc 4140caattagaag cagcaaagcg aaacagccca agaaaaaggt cggcccgtcg gccttttctg 4200caacgctgat cacgggcagc gatccaacca acaccctcca gagtgactag gggcggaaat 4260ttatcgggat taatttccac tcaaccacaa atcacagtcg tccccggtaa tttaacggct 4320gcagacggca atttaacggc ttctgcgaat cgcttggatt ccccgcccct ggccgtagag 4380cttaaagtat gtcccttgtc gatgcgatgt atgaattcat ggtgttttga tcattttaaa 4440tttttatatg gcgggtggtg ggcaactcgc ttgcgcgggc aactcgctta ccgattacgt 4500tagggctgat atttacgtaa aaatcgtcaa gggatgcaag accaaaccgt taaatttccg 4560gagtcaacag catccaagcc caagtccttc acggagaaac cccagcgtcc acatcacgag 4620cgaaggacca cctctaggca tcggacgcac catccaatta gaagcagcaa agcgaaacag 4680cccaagaaaa aggtcggccc gtcggccttt tctgcaacgc tgatcacggg cagcgatcca 4740accaacaccc tccagagtga ctaggggcgg aaatttatcg ggattaattt ccactcaacc 4800acaaatcaca gtcgtccccg gtaatttaac ggctgcagac ggcaatttaa cggcttctgc 4860gaatcgcttg gattccccgc ccctggccgt agagcttaaa gtatgtccct tgtcgatgcg 4920atgtatcaca acatataaat actggcaagg gatgccatgc ttggagtttc caactcaatt 4980tacctctatc cacacttctc ttccttcctc aatcctctat atacacaact ggggatcctt 5040caccatgagg agctcccttg tgctgttctt tgtctctgcg tggacggcct tggctagccc 5100tattcgtcct cgaccggtct cgcaggatct gtttaaccag ttcaatctct ttgcacagta 5160ttcagctgcc gcatactgcg gaaaaaacaa tgatgcccca gcaggtacaa acattacgtg 5220cacgggaaat gcctgccccg aggtagagaa ggcggatgca acgtttctct actcgtttga 5280agactctgga gtgggcgatg tcaccggctt ccttgctctc gacaacacga acaaattgat 5340cgtcctctct ttccgtggct ctcgttccat agagaactgg atcgggaatc ttaacttcga 5400cttgaaaaaa ataaatgaca tttgctccgg ctgcagggga catgacggct tcacttcgtc 5460ctggaggtct gtagccgata cgttaaggca gaaggtggag gatgctgtga gggagcatcc 5520caactatcgc gtggtgttta ccggacatag cttgggtggt gcattggcaa ctgttgccgg 5580agcagacctg cgtggaaatg ggtatgatat cgacgtgttt tcatatggcg ccccccgagt 5640cggaaacagg gcttttgcag aattcctgac cgtacagacc ggcggaacac tctaccgcat 5700tacccacacc aatgatattg tccctagact cccgccgcgc gaattcggtt acagccattc 5760tagcccagaa tactggatca aatctggaac ccttgtcccc gtcacccgaa acgatatcgt 5820gaagatagaa ggcatcgatg ccaccggcgg caataaccgg ccgaacattc cggatatccc 5880tgcgcaccta tggtacttcg ggttaattgg gacatgtctt tagtgcgcgg cgcggctggg 5940tcgactctag cgagctcgag atctagaggg tgactgacac ctggcggtag acaatcaatc 6000catttcgcta tagttaaagg atggggatga gggcaattgg ttatatgatc atgtatgtag 6060tgggtgtgca taatagtagt gaaatggaag ccaagtcatg tgattgtaat cgaccgacgg 6120aattgaggat atccggaaat acagacaccg tgaaagccat ggtctttcct tcgtgtagaa 6180gaccagacag acagtccctg atttaccctt gcacaaagca ctagaaaatt agcattccat 6240ccttctctgc ttgctctgct gatatcactg tcattcaatg catagccatg agctcatctt 6300agatccaagc acgtaattcc atagccgagg tccacagtgg agcagcaaca ttccccatca 6360ttgctttccc caggggcctc ccaacgacta aatcaagagt atatctctac cgtccaatag 6420atcgtcttcg cttcaaaatc tttgacaatt ccaagagggt ccccatccat caaacccagt 6480tcaataatag ccgagatgca tggtggagtc aattaggcag tattgctgga atgtcgggcc 6540agttggccgg gtggtcattg gccgccagta cgactgtgat gccatctgcc actaaatccg 6600atcattgatc caccgcccac gaggcgcgtc tttgcttttt gcgcggcgtc caggttcaac 6660tctctcgctc tagatatcga tgaattcact ggccgtcgtt ttacaacgtc gtgactggga 6720aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 6780taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 6840atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg 6900gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc 6960aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 7020tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 7080gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt 7140ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 7200tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 7260ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 7320ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 7380tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 7440gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 7500gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat 7560acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 7620tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 7680caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 7740gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 7800cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 7860tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 7920agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 7980tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 8040ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 8100acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 8160ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 8220gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 8280gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 8340ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 8400gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 8460ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 8520cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 8580cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 8640ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 8700tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 8760cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 8820ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 8880aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 8940ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 9000tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 9060gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 9120gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg 9180caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct 9240tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta 9300tgaccatgat tacgccaagc t 9321198775DNAArtificial Sequencecomplete DNA sequence of plasmid pMT2786 19tgtataagct agcttatggt gttttgatca ttttaaattt ttatatggcg ggtggtgggc 60aactcgcttg cgcgggcaac tcgcttaccg attacgttag ggctgatatt tacgtaaaaa 120tcgtcaaggg atgcaagacc aaaccgttaa atttccggag tcaacagcat ccaagcccaa 180gtccttcacg gagaaacccc agcgtccaca tcacgagcga aggaccacct ctaggcatcg 240gacgcaccat ccaattagaa gcagcaaagc gaaacagccc aagaaaaagg tcggcccgtc 300ggccttttct gcaacgctga tcacgggcag cgatccaacc aacaccctcc agagtgacta 360ggggcggaaa tttatcggga ttaatttcca ctcaaccaca aatcacagtc gtccccggta 420atttaacggc tgcagacggc aatttaacgg cttctgcgaa tcgcttggat tccccgcccc 480tggccgtaga gcttaaagta tgtcccttgt cgatgcgatg tatgaattca tggtgttttg 540atcattttaa atttttatat ggcgggtggt gggcaactcg cttgcgcggg caactcgctt 600accgattacg ttagggctga tatttacgta aaaatcgtca agggatgcaa gaccaaaccg 660ttaaatttcc ggagtcaaca gcatccaagc ccaagtcctt cacggagaaa ccccagcgtc 720cacatcacga gcgaaggacc acctctaggc atcggacgca ccatccaatt agaagcagca 780aagcgaaaca gcccaagaaa aaggtcggcc cgtcggcctt ttctgcaacg ctgatcacgg 840gcagcgatcc aaccaacacc ctccagagtg actaggggcg gaaatttatc gggattaatt 900tccactcaac cacaaatcac agtcgtcccc ggtaatttaa cggctgcaga cggcaattta 960acggcttctg cgaatcgctt ggattccccg cccctggccg tagagcttaa agtatgtccc 1020ttgtcgatgc gatgtatcac aacatataaa tactggcaag ggatgccatg cttggagttt 1080ccaactcaat ttacctctat ccacacttct cttccttcct caatcctcta tatacacaac 1140tggggatcca caatgaagtg ggtaaccttt atttcccttc tttttctctt tagctcggct 1200tattccaggg gtgtgtttcg tcgagatgca cacaagagtg aggttgctca tcggtttaaa 1260gatttgggag aagaaaattt caaagccttg gtgttgattg cctttgctca gtatcttcag 1320cagtgtccat ttgaagatca tgtaaaatta gtgaatgaag taactgaatt tgcaaaaaca 1380tgtgttgctg atgagtcagc tgaaaattgt gacaaatcac ttcataccct ttttggagac 1440aaattatgca cagttgcaac tcttcgtgaa acctatggtg aaatggctga ctgctgtgca 1500aaacaagaac ctgagagaaa tgaatgcttc ttgcaacaca aagatgacaa cccaaacctc 1560ccccgattgg tgagaccaga ggttgatgtg atgtgcactg cttttcatga caatgaagag 1620acatttttga aaaaatactt atatgaaatt gccagaagac atccttactt ttatgccccg 1680gaactccttt tctttgctaa aaggtataaa gctgctttta cagaatgttg ccaagctgct 1740gataaagctg cctgcctgtt gccaaagctc gatgaacttc gggatgaagg gaaggcttcg 1800tctgccaaac agagactcaa gtgtgccagt ctccaaaaat ttggagaaag agctttcaaa 1860gcatgggcag tagctcgcct gagccagaga tttcccaaag ctgagtttgc agaagtttcc 1920aagttagtga cagatcttac caaagtccac acggaatgct gccatggaga tctgcttgaa 1980tgtgctgatg acagggcgga ccttgccaag tatatctgtg aaaatcaaga ttcgatctcc 2040agtaaactga aggaatgctg tgaaaaacct ctgttggaaa aatcccactg cattgccgaa 2100gtggaaaatg atgagatgcc tgctgacttg ccttcattag ctgctgattt tgttgaaagt 2160aaggatgttt gcaaaaacta tgctgaggca aaggatgtct tcctgggcat gtttttgtat 2220gaatatgcaa gaaggcatcc tgattactct gtcgtgctgc tgctgagact tgccaagaca 2280tatgaaacca ctctagagaa gtgctgtgcc gctgcagatc ctcatgaatg ctatgccaaa 2340gtgttcgatg aatttaaacc tcttgtggaa gagcctcaga atttaatcaa acaaaattgt 2400gagctttttg agcagcttgg agagtacaaa ttccagaatg cgctattagt tcgttacacc 2460aagaaagtac cccaagtgtc aactccaact cttgtagagg tctcaagaaa cctaggaaaa 2520gtgggcagca aatgttgtaa acatcctgaa gcaaaaagaa tgccctgtgc agaagactat 2580ctatccgtgg tcctgaacca gttatgtgtg ttgcatgaga aaacgccagt aagtgacaga 2640gtcaccaaat gctgcacaga atccttggtg aacaggcgac catgcttttc agctctggaa 2700gtcgatgaaa catacgttcc caaagagttt aatgctgaaa cattcacctt ccatgcagat 2760atatgcacac tttctgagaa ggagagacaa atcaagaaac aaactgcact tgttgagctc 2820gtgaaacaca agcccaaggc aacaaaagag caactgaaag ctgttatgga tgatttcgca 2880gcttttgtag agaagtgctg caaggctgac gataaggaga cctgctttgc cgaggagggt 2940aaaaaacttg ttgctgcaag tcaagctgcc ttaggcttat aactcgagat ctagagggtg 3000actgacacct ggcggtagac aatcaatcca tttcgctata gttaaaggat ggggatgagg 3060gcaattggtt atatgatcat gtatgtagtg ggtgtgcata atagtagtga aatggaagcc 3120aagtcatgtg attgtaatcg accgacggaa ttgaggatat ccggaaatac agacaccgtg 3180aaagccatgg tctttccttc gtgtagaaga ccagacagac agtccctgat ttacccttgc 3240acaaagcact agaaaattag cattccatcc ttctctgctt gctctgctga tatcactgtc 3300attcaatgca tagccatgag ctcatcttag atccaagcac gtaattccat agccgaggtc 3360cacagtggag cagcaacatt ccccatcatt gctttcccca ggggcctccc aacgactaaa 3420tcaagagtat atctctaccg tccaatagat cgtcttcgct tcaaaatctt tgacaattcc 3480aagagggtcc ccatccatca aacccagttc aataatagcc gagatgcatg gtggagtcaa 3540ttaggcagta ttgctggaat gtcggggcca gttggccggg tggtcattgg ccgcctgtga 3600tgccatctgc cactaaatcc gatcattgat ccaccgccca cgaggcgcgt ctttgctttt 3660tgcgcggcgt ccaggttcaa ctctctcctc tagactggaa acgcaaccct gaagggattc 3720ttcctttgag agatggaagc gtgtcatatc tcttcggttc tacggcaggt ttttttctgc 3780tctttcgtag catggcatgg tcacttcagc gcttatttac agttgctggt attgatttct 3840tgtgcaaatt gctatctgac acttattagc tatggagtca ccacatttcc cagcaacttc 3900cccacttcct ctgcaatcgc caacgtcctc tcttcactga gtctccgtcc gataacctgc 3960actgcaaccg gtgccccatg gtacgcctcc ggatcatact cttcctgcac gagggcatca 4020agctcactaa ccgccttgaa actctcattc ttcttatcga tgttcttatc cgcaaaggta 4080accggaacaa ccacgctcgt gaaatccagc aggttgatca cagaggcata cccatagtac 4140cggaactggt catgccgtac cgcagcggta ggcgtaatcg gcgcgatgat ggcgtccagt 4200tccttcccgg ccttttcttc agcctcccgc catttctcaa ggtactccat ctggtaattc 4260cacttctgga gatgcgtgtc ccagagctcg ttcatgttaa cagctttgat gttcgggttc 4320agtaggtctt tgatatttgg aatcgccggc tcgccggatg cactgatatc gcgcattacg 4380tcggcgctgc cgtcagccgc gtagatatgg gagatgagat cgtggccgaa atcgtgcttg 4440tatggcgtcc acggggtcac ggtgtgaccg gctttggcga gtgcggcgac ggtggtttcc 4500acgccgcgca ggataggagg gtgtggaagg acattgccgt cgaagttgta gtagccgata 4560ttgagcccgc cgttcttgat cttggaggca ataatgtccg actcggactg gcgccagggc 4620atggggatga ccttggagtc gtatttccat ggctcctgac cgaggacgga tttggtgaag 4680aggcggaggt ctaacatact tcatcagtga ctgccggtct cgtatatagt ataaaaagca 4740agaaaggagg acagtggagg cctggtatag agcaggaaaa gaaggaagag gcgaaggact 4800caccctcaac agagtgcgta atcggcccga caacgctgtg caccgtctcc tgaccctcca 4860tgctgttcgc catctttgca tacggcagcc gcccatgact cggccttaga ccgtacagga 4920agttgaacgc ggccggcact

cgaatcgagc caccgatatc cgttcctaca ccgatgacgc 4980caccacgaat cccaacgatc gcaccctcac caccagaact gccgccgcac gaccagttct 5040tgttgcgtgg gttgacggtg cgcccgatga tgttgttgac tgtctcgcag accatcaggg 5100tctgcgggac agaggtcttg acgtagaaga cggcaccggc tttgcggagc atggttgtca 5160gaaccgagtc cccttcgtcg tacttgttta gccatgagat gtagcccatt gatgtttcgt 5220agccctggtg gcatatgtta gctgacaaaa agggacatct aacgacttag gggcaacggt 5280gtaccttgac tcgaagctgg tctttgagag agatggggag gccatggagt ggaccaacgg 5340gtctcttgtg ctttgcgtag tattcatcga gttcccttgc ctgcgcgaga gcggcgtcag 5400ggaagaactc gtgggcgcag tttgtctgca cagaagccag cgtcagcttg atagtcccat 5460aaggtggcgt tgttacatct ccctgagagg tagaggggac cctactaact gctgggcgat 5520tgctgcccgt ttacagaatg ctagcgtaac ttccaccgag gtcaactctc cggccgccag 5580cttggacaca agatctgcag cggaggcctc tgtgatcttc agttcggcct ctgaaaggat 5640caccgatttc tttgggaaat caataacgct gtcttccgca ggcagcgtct ggactttcca 5700ttcatcaggg atggtttttg cgaggcgggc gcgcttatca gcggccagtt cttcccagga 5760ttgaggcatt ctgtgttagc ttatagtcag gatgttggct cgacgagtgt aaactgggag 5820ttggcatgag ggttatgtag gcttctttag ccccgcatcc ccctcattct cctcattgat 5880cccgggggag cggatggtgt tgataagaga ctaattatag ggtttagctg gtgcctagct 5940ggtgattggc tggcttcgcc gaattttacg ggccaaggaa agctgcagaa ccgcggcact 6000ggtaaacggt aattaagcta tcagccccat gctaacgagt ttaaattacg tgtattgctg 6060ataaacacca acagagcttt actgaaagat gggagtcacg gtgtggcttc cccactgcga 6120ttattgcaca agcagcgagg gcgaacttga ctgtcgtcgc tgagcagcct gcagtcaaac 6180atacatatat atcaaccgcg aagacgtctg gccttgtaga acacgacgct ccctagcaac 6240acctgccgtg tcagcctcta cggttgttac ttgcattcag gatgctctcc agcgggcgag 6300ctattcaaaa tattcaaagc aggtatctcg tattgccagg attcagctga agcaacaggt 6360gccaaggaaa tctgcgtcgg ttctcatctg ggcttgctcg gtcctggcgt agatctagag 6420tcgacctgca ggcatgcggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 6480ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 6540taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 6600aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 6660attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 6720cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 6780gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 6840ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 6900agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 6960tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 7020ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 7080gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 7140ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 7200gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 7260aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 7320aagccagtta ccttcggaaa aagagttggt agctcttgat ccgacaaaca aaccaccgct 7380ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 7440gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 7500gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 7560tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaagcaagg 7620attttcttaa cttcttcggc gacagcatca ccgacttcgg tggtactgtt ggaaccacct 7680aaatcaccag ttctgatacc tgcatccaaa acctttttaa ctgcatcttc aatggcctta 7740ccttcttcag gcaagttcaa tgacaatttc aacatcattg cagcagacaa gatagtggcg 7800atagggttga ccttattctt tggcaaatct ggagcagaac cgtggcatgg ttcgtacaaa 7860ccaaatgcgg tgttcttgtc tggcaaagag gccaaggacg cagatggcaa caaacccaag 7920gaacctggga taacggaggc ttcatcggag atgatatcac caaacatgtt gctggtgatt 7980ataataccat ttaggtgggt tgggttctta actaggatca tggcggcaga atcaatcaat 8040tgatgttgaa ccttcaatgt agggaattcg ttcttgatgg tttcctccac agtttttctc 8100cataatcttg aagaggccaa aacattagct ttatccaagg accaaatagg caatggtggc 8160tcatgttgta gggccatgaa agcggccatt cttgtgattc tttgcacttc tggaacggtg 8220tattgttcac tatcccaagc gacaccatca ccatcgtctt cctttctctt accaaagtaa 8280atacctccca ctaattctct gacaacaacg aagtcagtac ctttagcaaa ttgtggcttg 8340attggagata agtctaaaag agagtcggat gcaaagttac atggtcttaa gttggcgtac 8400aattgaagtt ctttacggat ttttagtaaa ccttgttcag gtctaacact gccggtaccc 8460catttaggac cacccacagc acctaacaaa acggcatcag ccttcttgga ggcttccagc 8520gcctcatctg gaagtggaac acctgtagca tcgatagcag caccaccaat taaatgattt 8580tcgaaatcga acttgacatt ggaacgaaca tcagaaatag ctttaagaac cttaatggct 8640tcggctgtga tttcttgacc aacgtggtca cctggcaaaa cgacgatctt cttaggggca 8700gacatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 8760ggatacatat ttgaa 8775205857DNAArtificial Sequencecomplete DNA sequence of plasmid pMT3155 20tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 60gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 120ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 180cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 240caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 300ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 360taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 420taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 480cttcggaaaa agagttggta gctcttgatc cgacaaacaa accaccgctg gtagcggtgg 540tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 600gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 660catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 720atcaatctaa agtatatatg agtaaacttg gtctgacagt taagcaagga ttttcttaac 780ttcttcggcg acagcatcac cgacttcggt ggtactgttg gaaccaccta aatcaccagt 840tctgatacct gcatccaaaa cctttttaac tgcatcttca atggccttac cttcttcagg 900caagttcaat gacaatttca acatcattgc agcagacaag atagtggcga tagggttgac 960cttattcttt ggcaaatctg gagcagaacc gtggcatggt tcgtacaaac caaatgcggt 1020gttcttgtct ggcaaagagg ccaaggacgc agatggcaac aaacccaagg aacctgggat 1080aacggaggct tcatcggaga tgatatcacc aaacatgttg ctggtgatta taataccatt 1140taggtgggtt gggttcttaa ctaggatcat ggcggcagaa tcaatcaatt gatgttgaac 1200cttcaatgta gggaattcgt tcttgatggt ttcctccaca gtttttctcc ataatcttga 1260agaggccaaa acattagctt tatccaagga ccaaataggc aatggtggct catgttgtag 1320ggccatgaaa gcggccattc ttgtgattct ttgcacttct ggaacggtgt attgttcact 1380atcccaagcg acaccatcac catcgtcttc ctttctctta ccaaagtaaa tacctcccac 1440taattctctg acaacaacga agtcagtacc tttagcaaat tgtggcttga ttggagataa 1500gtctaaaaga gagtcggatg caaagttaca tggtcttaag ttggcgtaca attgaagttc 1560tttacggatt tttagtaaac cttgttcagg tctaacactg ccggtacccc atttaggacc 1620acccacagca cctaacaaaa cggcatcagc cttcttggag gcttccagcg cctcatctgg 1680aagtggaaca cctgtagcat cgatagcagc accaccaatt aaatgatttt cgaaatcgaa 1740cttgacattg gaacgaacat cagaaatagc tttaagaacc ttaatggctt cggctgtgat 1800ttcttgacca acgtggtcac ctggcaaaac gacgatcttc ttaggggcag acatactctt 1860cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 1920tgaatgtata agctagagcg ctgtcgacac tagtagatct cgatcgcgta cggctagcaa 1980gctttggcca cctaggctag aaagaaggat tacctctaaa caagtgtacc tgtgcattct 2040gggtaaacga ctcataggag agttgtaaaa aagtttcggc cggcgtattg ggtgttacgg 2100agcattcact aggcaaccat gcatggttac tattgtatac ccatcttagt aggaatgatt 2160ttcgaggttt atacctacga tgaatgtgtg tcctgtaggc ttgagagttc aaggaagaaa 2220cagtgcaatt atctttgcga acccaggggc tggtgacgga attttcatag tcaagctatc 2280agagtaaaga agaggagcat gtcaaagtac aattagagac aaatatatag tcgcgtggag 2340ccaagagcgg attcctcagt ctcgtaggtc tcttgacgac cgttgatctg cttgatctcg 2400tctcccgaaa atgaaaatag actctgctaa gctattcttc tgcttcgccg gagcctgaag 2460ggcgtactag ggttgcgagg tccaatgcat taatgcattg cagatgagct gtatctggaa 2520gaggtaaacc cgaaacgcgt tttattcttg ttgacatgga gctattaaat cactagaagg 2580cactctttgc tgcttggaca aatgaacgta tcttatcgag atcctgaaca ccatttgtct 2640caactccgga gctgacatcg acaccaacga tcttatatcc agattcgtca agctgtttga 2700tgatttcagt aacgttaagt ggatcgatcc ggatagcgcg ggttccttcc ggtattgtct 2760ccttccgtgt ttcagttagc ctcccccatc tcccgggcaa acgtgcgcgc tcagatctcg 2820gtgacgggca ggaccggacg gggcggtacc ggcaggctga agtccagctg ccagaaaccc 2880acgtcatgcc agttcccgtg cttgaagccg gccgcccgca gcatgccgcg gggggcatat 2940ccgagcgcct cgtgcatgcg cacgctcggg tcgttgggca gcccgatgac agcgaccacg 3000ctcttgaagc cctgtgcctc cagggacttc agcaggtggg tgtagagcgt ggagcccagt 3060cccgtccgct ggtggcgggg ggagacgtac acggtcgact cggccgtcca gtcgtaggcg 3120ttgcgtgcct tccaggggcc cgcgtaggcg atgccggcga cctcgccgtc cacctcggcg 3180acgagccagg gatagcgctc ccgcagacgg acgaggtcgt ccgtccactc ctgcggttcc 3240tgcggctcgg tacggaagtt gaccgtgctt gtctcgatgt agtggttgac gatggtgcag 3300accgccggca tgtccgcctc ggtggcacgg cggatgtcgg ccgggcgtcg ttctgggctc 3360atggatccgg tggaagtaag atacgacgag tttgattgag aaaagacagg gtgattgtca 3420agttcagtat ggaagaaaga gtagaagaag atcagacgac agggaagagc gatgacataa 3480aaggtggaag acggaagaaa aacgaaccaa atcaatccca ctctatggcg ggggttggac 3540tgcctgaggc cggcactggt ggggcttatc gataagttct cgtcaccgga tgcaatgcgc 3600tgtcaactgc tgacttggcc ctgaacatcc tgtcctctac agatccatac tatacaatga 3660tcccagttat agtgcggtaa ggtgcatatc atatctcatt ctcatgactc attcgacttt 3720tttttagaga aaagtacata cgtggaacat acactaaacg caacaggtcg cgacaacact 3780ggtatacaaa acggtccccg gtgaatgacg ttattagtgt ctatccccca ctcacacccg 3840aaaagaataa tagaaactaa cagaaaaagc ggcccgagga taagaggaac attcaaacag 3900aaggggaatc ataaaaaccg aaaaatgcaa ggaaaagaga actcaaatca ataattttca 3960taatactgtc gagagtaata cggaccagcg tctctcaggg acatgcgtcg gcgcaaggca 4020tcatccaatc tctcatctaa cacatccagc attcgtgttc gatagtctaa ctgcttctct 4080cggcgctcaa gtcttgcttc ccgatcatcg agaattcggg tacggatatc gttgcgagct 4140cgtggcccac gatatctttc gtcgaactcg ggtgcatcca aatcctcgga actaacaggg 4200gcgtggataa tcttcgggcg atctcgggtc tgccgagcaa cttctcggct atggaccctc 4260catagttgct tgtccgaatc cctgctgtcg gcggggatca catcaacata gtcacttggg 4320tacttggtac ggccgtgtct gttgtcagat gcacccgagg gttgtttccg gtagtgggag 4380cgatatcgag atggcccagg acgccttgaa gtagaggaat gccggcgaaa gagacttccg 4440cgccagggct gcgcttcttt gcgtgtctct ccctcatgtt cagagtcatc tgtaactgag 4500ctaacgtctt caaaatcgaa aatctctgaa tcgtcatccc caatactgct ggagtcgggg 4560gccaactcta actctggttc cgccatgttg ggtggtttga cactacctcc ctgggtgaca 4620ttctcaaagg aatgagattc atacgggtgg aatggctctt tccgtgggtt gttattggtt 4680accgacaccc cggctgggtt gtacacgtta ttgacccctg ggtgttgcgc accagcactg 4740ggaacgcctc tggtgtgctc ctttagcacc tcaatagctg ggccattctc cggagcagcc 4800cttgcggtgt ggggcatggc tacctgtggg cgctggtgcg cacccacagg gacccctcta 4860acatgctcct tcaggacctc agcactgggg ccgttctctg gagcagctct ggagacgtga 4920ggtatgtgct gttgtacact accgctggca acacctctgg catgttcctt tagaacatca 4980atcgcgggtc ccttctctgg cgcggcccgc gcgacgtggg gcatggctgc gtgtgggtgt 5040accccgaaag agaaatgatc tggaacttct tgtggtagat gtccacggtt gaccaatggt 5100cttcctcccg cctgctgtgc ctgaggttga ggagggggag gcggcggtgg cggggcttgc 5160tgagtgaact cagggttaag ctgccgtggg atattgtgca cctgcaccat ggggggttgt 5220tgtgcatgac ccatattggg ccttgcccac tgaggggcgt tatcaacctc cgtgtccaat 5280ggccgaggat catcgatgac cacgggattt tcggccttcg gctcagcccg cggctcaatt 5340ttctccttag gcttgctagg taaatcgaac tccacttgcg ctactttggg acgcgaagag 5400gtgataggct tccccatgat ctagagtcga cctgcaggca tgcggcgtaa tcatggtcat 5460agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 5520gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 5580gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 5640aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 5700cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 5760ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 5820aggccaggaa ccgtaaaaag gccgcgttgc tggcgtt 5857217860DNAArtificial Sequencecomplete DNA sequence of plasmid pMT3296 21tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 60gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 120ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 180cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 240caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 300ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 360taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 420taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 480cttcggaaaa agagttggta gctcttgatc cgacaaacaa accaccgctg gtagcggtgg 540tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 600gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 660catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 720atcaatctaa agtatatatg agtaaacttg gtctgacagt taagcaagga ttttcttaac 780ttcttcggcg acagcatcac cgacttcggt ggtactgttg gaaccaccta aatcaccagt 840tctgatacct gcatccaaaa cctttttaac tgcatcttca atggccttac cttcttcagg 900caagttcaat gacaatttca acatcattgc agcagacaag atagtggcga tagggttgac 960cttattcttt ggcaaatctg gagcagaacc gtggcatggt tcgtacaaac caaatgcggt 1020gttcttgtct ggcaaagagg ccaaggacgc agatggcaac aaacccaagg aacctgggat 1080aacggaggct tcatcggaga tgatatcacc aaacatgttg ctggtgatta taataccatt 1140taggtgggtt gggttcttaa ctaggatcat ggcggcagaa tcaatcaatt gatgttgaac 1200cttcaatgta gggaattcgt tcttgatggt ttcctccaca gtttttctcc ataatcttga 1260agaggccaaa acattagctt tatccaagga ccaaataggc aatggtggct catgttgtag 1320ggccatgaaa gcggccattc ttgtgattct ttgcacttct ggaacggtgt attgttcact 1380atcccaagcg acaccatcac catcgtcttc ctttctctta ccaaagtaaa tacctcccac 1440taattctctg acaacaacga agtcagtacc tttagcaaat tgtggcttga ttggagataa 1500gtctaaaaga gagtcggatg caaagttaca tggtcttaag ttggcgtaca attgaagttc 1560tttacggatt tttagtaaac cttgttcagg tctaacactg ccggtacccc atttaggacc 1620acccacagca cctaacaaaa cggcatcagc cttcttggag gcttccagcg cctcatctgg 1680aagtggaaca cctgtagcat cgatagcagc accaccaatt aaatgatttt cgaaatcgaa 1740cttgacattg gaacgaacat cagaaatagc tttaagaacc ttaatggctt cggctgtgat 1800ttcttgacca acgtggtcac ctggcaaaac gacgatcttc ttaggggcag acatactctt 1860cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 1920tgaatgtata agctagagcg ctgtcgacac tagtagatct cgatcgcgta ccctcagcaa 1980agccatcatt gcatgggatg gtcaaatcga cacgcgaaat ccaaggagag aacccgccat 2040gactagcaga gagccctgcg ctcagatgaa tcgcaaagcc cgaggattgg tatcagcgaa 2100agtatcatcc acaattttgt ctaccgactt gacatagata gtcccttccg atgaatatat 2160tccacctctc ccaaccactg aggctaatat gcagttatca acaatccacg caaaccagct 2220tgcattagac ccttctgctt ggataacatc cttgtttggc aggatttatc tgataggttc 2280aatatagata ctagacttct ataattattc ggagtgatcc aaagctgatt cctatcttat 2340cacccatgcc gtccgacgta tactgccaaa gattataatc agagatgaaa tgttggaaaa 2400tagagctgag cattctgcat ctggtcgttg tggtctgtaa ccttggtgag gccccgtcta 2460aaggaacgga tgctacacaa aactatggtt tgttagcatc caatatgtcg catctcttcc 2520cactactttg attcaaacca tggtacttgg ccgcatgacg taaaatagac ccgctctgga 2580tcaagattag tctaaagctt gtgatagggg gttacctttg taaaagacag gtgtcatggt 2640aatactatgg gcgaatatca gtcaaggtat cctgctcagc ctcacccctt gcctttttgg 2700gaagacttat agcaccctgc agattatcca ctgtgtaaat caacaagaat taatgacgct 2760ttgcatctta ctcgtggttc agtagtgata tacgtgtctt tctgtttgac taccctacat 2820tgctatgcag gcctagaccc acccccctga ggacttgcgt tatcctccta gaaattgatc 2880tgcctagcat atggactcat agttatgtta gttccagaaa tcgctgctgg aattgaatcc 2940ctgctcacta gtgccaacag agaaaaactg gaatgaacca atgagtaaat gctacgtaca 3000aagaatgtat ttaacaccta tgatgtatga atcgactgtg tccatatcta gggatatttc 3060tgctcagatg caaacctcgt ctcacttctg acaacctaat ccctctttta ccggtattta 3120tacatcgtat cccaaagtca agtacgtcag gtccgacgtc ctatccactc tacctgcaaa 3180atattaacct cacctctaat agaggaagac catatcccac catgagactc tccctcaacc 3240ttctactgat cgtcggatct gccgcagtcg cccgggctgc tctggtcccg gtcccggggg 3300ctagcgaaga actttgcgga cgtcttggag tcatgtatta cgatcctgat aatctccccg 3360agggtgtgga ggtgcatgag atacggaagt gtgccggaca tcccatgggt cgcgagaact 3420attggggctt gggtgattat ctgccgaggt ggtttcctta ggtattcttt caaagataac 3480atggtccaat gaacggtcta tgatatgggt ctgagacacc ggatctcaat ggttgcctat 3540agaccgtagg aaggatactt catgtacatg tcactggcta ctagtcactc attgcatttc 3600agtagtaacc cccgaaatag ataagaaagc atggtcgtgc agtattagta tatttaggaa 3660aaggtcatta atcattacca acacacttgc gttgagtcat cttactcaac cggcctcctt 3720ccccacacaa catgaaactt cccatagata tgcaccctag gatctctcaa ctcattccgc 3780acagaggcca agaaaactgc tacctccacc ggggaccagc cgagaaccct tgtacaaagc 3840gcaagagcat acgactcaag actcataacc atgacttcgc gatggaaccg accggcttct 3900tttaatcgct tttctttagc ccagggaccc atcgggaact acagaaatta agttaatacc 3960cgatagatta acggagtatt aaaccagagg gagtctaggc tagaaagaag gattacctct 4020aaacaagtgt acctgtgcat tctgggtaaa cgactcatag gagagttgta aaaaagtttc 4080ggccggcgta ttgggtgtta cggagcattc actaggcaac catgcatggt tactattgta 4140tacccatctt agtaggaatg attttcgagg tttataccta cgatgaatgt gtgtcctgta 4200ggcttgagag ttcaaggaag aaacagtgca attatctttg cgaacccagg ggctggtgac 4260ggaattttca tagtcaagct atcagagtaa agaagaggag catgtcaaag tacaattaga 4320gacaaatata tagtcgcgtg gagccaagag cggattcctc agtctcgtag gtctcttgac 4380gaccgttgat ctgcttgatc tcgtctcccg aaaatgaaaa tagactctgc taagctattc 4440ttctgcttcg ccggagcctg aagggcgtac tagggttgcg aggtccaatg cattaatgca 4500ttgcagatga gctgtatctg gaagaggtaa acccgaaacg cgttttattc ttgttgacat 4560ggagctatta aatcactaga aggcactctt tgctgcttgg acaaatgaac gtatcttatc 4620gagatcctga acaccatttg tctcaactcc ggagctgaca tcgacaccaa cgatcttata 4680tccagattcg tcaagctgtt tgatgatttc agtaacgtta agtggatcga tccggatagc 4740gcgggttcct tccggtattg tctccttccg tgtttcagtt agcctccccc atctcccggg 4800caaacgtgcg cgctcagatc tcggtgacgg gcaggaccgg acggggcggt accggcaggc 4860tgaagtccag ctgccagaaa cccacgtcat gccagttccc gtgcttgaag ccggccgccc 4920gcagcatgcc gcggggggca tatccgagcg cctcgtgcat gcgcacgctc gggtcgttgg 4980gcagcccgat gacagcgacc acgctcttga agccctgtgc ctccagggac ttcagcaggt 5040gggtgtagag cgtggagccc agtcccgtcc gctggtggcg gggggagacg tacacggtcg 5100actcggccgt ccagtcgtag gcgttgcgtg ccttccaggg gcccgcgtag gcgatgccgg 5160cgacctcgcc gtccacctcg

gcgacgagcc agggatagcg ctcccgcaga cggacgaggt 5220cgtccgtcca ctcctgcggt tcctgcggct cggtacggaa gttgaccgtg cttgtctcga 5280tgtagtggtt gacgatggtg cagaccgccg gcatgtccgc ctcggtggca cggcggatgt 5340cggccgggcg tcgttctggg ctcatggatc cggtggaagt aagatacgac gagtttgatt 5400gagaaaagac agggtgattg tcaagttcag tatggaagaa agagtagaag aagatcagac 5460gacagggaag agcgatgaca taaaaggtgg aagacggaag aaaaacgaac caaatcaatc 5520ccactctatg gcgggggttg gactgcctga ggccggcact ggtggggctt atcgataagt 5580tctcgtcacc ggatgcaatg cgctgtcaac tgctgacttg gccctgaaca tcctgtcctc 5640tacagatcca tactatacaa tgatcccagt tatagtgcgg taaggtgcat atcatatctc 5700attctcatga ctcattcgac ttttttttag agaaaagtac atacgtggaa catacactaa 5760acgcaacagg tcgcgacaac actggtatac aaaacggtcc ccggtgaatg acgttattag 5820tgtctatccc ccactcacac ccgaaaagaa taatagaaac taacagaaaa agcggcccga 5880ggataagagg aacattcaaa cagaagggga atcataaaaa ccgaaaaatg caaggaaaag 5940agaactcaaa tcaataattt tcataatact gtcgagagta atacggacca gcgtctctca 6000gggacatgcg tcggcgcaag gcatcatcca atctctcatc taacacatcc agcattcgtg 6060ttcgatagtc taactgcttc tctcggcgct caagtcttgc ttcccgatca tcgagaattc 6120gggtacggat atcgttgcga gctcgtggcc cacgatatct ttcgtcgaac tcgggtgcat 6180ccaaatcctc ggaactaaca ggggcgtgga taatcttcgg gcgatctcgg gtctgccgag 6240caacttctcg gctatggacc ctccatagtt gcttgtccga atccctgctg tcggcgggga 6300tcacatcaac atagtcactt gggtacttgg tacggccgtg tctgttgtca gatgcacccg 6360agggttgttt ccggtagtgg gagcgatatc gagatggccc aggacgcctt gaagtagagg 6420aatgccggcg aaagagactt ccgcgccagg gctgcgcttc tttgcgtgtc tctccctcat 6480gttcagagtc atctgtaact gagctaacgt cttcaaaatc gaaaatctct gaatcgtcat 6540ccccaatact gctggagtcg ggggccaact ctaactctgg ttccgccatg ttgggtggtt 6600tgacactacc tccctgggtg acattctcaa aggaatgaga ttcatacggg tggaatggct 6660ctttccgtgg gttgttattg gttaccgaca ccccggctgg gttgtacacg ttattgaccc 6720ctgggtgttg cgcaccagca ctgggaacgc ctctggtgtg ctcctttagc acctcaatag 6780ctgggccatt ctccggagca gcccttgcgg tgtggggcat ggctacctgt gggcgctggt 6840gcgcacccac agggacccct ctaacatgct ccttcaggac ctcagcactg gggccgttct 6900ctggagcagc tctggagacg tgaggtatgt gctgttgtac actaccgctg gcaacacctc 6960tggcatgttc ctttagaaca tcaatcgcgg gtcccttctc tggcgcggcc cgcgcgacgt 7020ggggcatggc tgcgtgtggg tgtaccccga aagagaaatg atctggaact tcttgtggta 7080gatgtccacg gttgaccaat ggtcttcctc ccgcctgctg tgcctgaggt tgaggagggg 7140gaggcggcgg tggcggggct tgctgagtga actcagggtt aagctgccgt gggatattgt 7200gcacctgcac catggggggt tgttgtgcat gacccatatt gggccttgcc cactgagggg 7260cgttatcaac ctccgtgtcc aatggccgag gatcatcgat gaccacggga ttttcggcct 7320tcggctcagc ccgcggctca attttctcct taggcttgct aggtaaatcg aactccactt 7380gcgctacttt gggacgcgaa gaggtgatag gcttccccat gatctagagt cgacctgcag 7440gcatgcggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 7500tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 7560ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 7620ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 7680ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 7740agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 7800catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 7860227185DNAArtificial Sequencecomplete DNA sequence of plasmid pMT3306 22tgtataagct agcttatggt gttttgatca ttttaaattt ttatatggcg ggtggtgggc 60aactcgcttg cgcgggcaac tcgcttaccg attacgttag ggctgatatt tacgtaaaaa 120tcgtcaaggg atgcaagacc aaaccgttaa atttccggag tcaacagcat ccaagcccaa 180gtccttcacg gagaaacccc agcgtccaca tcacgagcga aggaccacct ctaggcatcg 240gacgcaccat ccaattagaa gcagcaaagc gaaacagccc aagaaaaagg tcggcccgtc 300ggccttttct gcaacgctga tcacgggcag cgatccaacc aacaccctcc agagtgacta 360ggggcggaaa tttatcggga ttaatttcca ctcaaccaca aatcacagtc gtccccggta 420atttaacggc tgcagacggc aatttaacgg cttctgcgaa tcgcttggat tccccgcccc 480tggccgtaga gcttaaagta tgtcccttgt cgatgcgatg tatgaattca tggtgttttg 540atcattttaa atttttatat ggcgggtggt gggcaactcg cttgcgcggg caactcgctt 600accgattacg ttagggctga tatttacgta aaaatcgtca agggatgcaa gaccaaaccg 660ttaaatttcc ggagtcaaca gcatccaagc ccaagtcctt cacggagaaa ccccagcgtc 720cacatcacga gcgaaggacc acctctaggc atcggacgca ccatccaatt agaagcagca 780aagcgaaaca gcccaagaaa aaggtcggcc cgtcggcctt ttctgcaacg ctgatcacgg 840gcagcgatcc aaccaacacc ctccagagtg actaggggcg gaaatttatc gggattaatt 900tccactcaac cacaaatcac agtcgtcccc ggtaatttaa cggctgcaga cggcaattta 960acggcttctg cgaatcgctt ggattccccg cccctggccg tagagcttaa agtatgtccc 1020ttgtcgatgc gatgtatcac aacatataaa tactggcaag ggatgccatg cttggagttt 1080ccaactcaat ttacctctat ccacacttct cttccttcct caatcctcta tatacacaac 1140tggggatcca ccatgagact ctccctcaac cttctactga tcgtcggatc tgccgcagtc 1200gcccgggctg ctctggtccc ggtcccgggg gctagcgaag aactttgcgg acgtcttgga 1260gtcatgtatt acgatcctga taatctcccc gagggtgtgg aggtgcatga gatacggaag 1320tgtgccggac atcccatggg tcgcgagaac tattggggct tgggtgatta tctgccgagg 1380tggtttcctt agctcgagat ctagagggtg actgacacct ggcggtagac aatcaatcca 1440tttcgctata gttaaaggat ggggatgagg gcaattggtt atatgatcat gtatgtagtg 1500ggtgtgcata atagtagtga aatggaagcc aagtcatgtg attgtaatcg accgacggaa 1560ttgaggatat ccggaaatac agacaccgtg aaagccatgg tctttccttc gtgtagaaga 1620ccagacagac agtccctgat ttacccttgc acaaagcact agaaaattag cattccatcc 1680ttctctgctt gctctgctga tatcactgtc attcaatgca tagccatgag ctcatcttag 1740atccaagcac gtaattccat agccgaggtc cacagtggag cagcaacatt ccccatcatt 1800gctttcccca ggggcctccc aacgactaaa tcaagagtat atctctaccg tccaatagat 1860cgtcttcgct tcaaaatctt tgacaattcc aagagggtcc ccatccatca aacccagttc 1920aataatagcc gagatgcatg gtggagtcaa ttaggcagta ttgctggaat gtcggggcca 1980gttggccggg tggtcattgg ccgcctgtga tgccatctgc cactaaatcc gatcattgat 2040ccaccgccca cgaggcgcgt ctttgctttt tgcgcggcgt ccaggttcaa ctctctcctc 2100tagactggaa acgcaaccct gaagggattc ttcctttgag agatggaagc gtgtcatatc 2160tcttcggttc tacggcaggt ttttttctgc tctttcgtag catggcatgg tcacttcagc 2220gcttatttac agttgctggt attgatttct tgtgcaaatt gctatctgac acttattagc 2280tatggagtca ccacatttcc cagcaacttc cccacttcct ctgcaatcgc caacgtcctc 2340tcttcactga gtctccgtcc gataacctgc actgcaaccg gtgccccatg gtacgcctcc 2400ggatcatact cttcctgcac gagggcatca agctcactaa ccgccttgaa actctcattc 2460ttcttatcga tgttcttatc cgcaaaggta accggaacaa ccacgctcgt gaaatccagc 2520aggttgatca cagaggcata cccatagtac cggaactggt catgccgtac cgcagcggta 2580ggcgtaatcg gcgcgatgat ggcgtccagt tccttcccgg ccttttcttc agcctcccgc 2640catttctcaa ggtactccat ctggtaattc cacttctgga gatgcgtgtc ccagagctcg 2700ttcatgttaa cagctttgat gttcgggttc agtaggtctt tgatatttgg aatcgccggc 2760tcgccggatg cactgatatc gcgcattacg tcggcgctgc cgtcagccgc gtagatatgg 2820gagatgagat cgtggccgaa atcgtgcttg tatggcgtcc acggggtcac ggtgtgaccg 2880gctttggcga gtgcggcgac ggtggtttcc acgccgcgca ggataggagg gtgtggaagg 2940acattgccgt cgaagttgta gtagccgata ttgagcccgc cgttcttgat cttggaggca 3000ataatgtccg actcggactg gcgccagggc atggggatga ccttggagtc gtatttccat 3060ggctcctgac cgaggacgga tttggtgaag aggcggaggt ctaacatact tcatcagtga 3120ctgccggtct cgtatatagt ataaaaagca agaaaggagg acagtggagg cctggtatag 3180agcaggaaaa gaaggaagag gcgaaggact caccctcaac agagtgcgta atcggcccga 3240caacgctgtg caccgtctcc tgaccctcca tgctgttcgc catctttgca tacggcagcc 3300gcccatgact cggccttaga ccgtacagga agttgaacgc ggccggcact cgaatcgagc 3360caccgatatc cgttcctaca ccgatgacgc caccacgaat cccaacgatc gcaccctcac 3420caccagaact gccgccgcac gaccagttct tgttgcgtgg gttgacggtg cgcccgatga 3480tgttgttgac tgtctcgcag accatcaggg tctgcgggac agaggtcttg acgtagaaga 3540cggcaccggc tttgcggagc atggttgtca gaaccgagtc cccttcgtcg tacttgttta 3600gccatgagat gtagcccatt gatgtttcgt agccctggtg gcatatgtta gctgacaaaa 3660agggacatct aacgacttag gggcaacggt gtaccttgac tcgaagctgg tctttgagag 3720agatggggag gccatggagt ggaccaacgg gtctcttgtg ctttgcgtag tattcatcga 3780gttcccttgc ctgcgcgaga gcggcgtcag ggaagaactc gtgggcgcag tttgtctgca 3840cagaagccag cgtcagcttg atagtcccat aaggtggcgt tgttacatct ccctgagagg 3900tagaggggac cctactaact gctgggcgat tgctgcccgt ttacagaatg ctagcgtaac 3960ttccaccgag gtcaactctc cggccgccag cttggacaca agatctgcag cggaggcctc 4020tgtgatcttc agttcggcct ctgaaaggat caccgatttc tttgggaaat caataacgct 4080gtcttccgca ggcagcgtct ggactttcca ttcatcaggg atggtttttg cgaggcgggc 4140gcgcttatca gcggccagtt cttcccagga ttgaggcatt ctgtgttagc ttatagtcag 4200gatgttggct cgacgagtgt aaactgggag ttggcatgag ggttatgtag gcttctttag 4260ccccgcatcc ccctcattct cctcattgat cccgggggag cggatggtgt tgataagaga 4320ctaattatag ggtttagctg gtgcctagct ggtgattggc tggcttcgcc gaattttacg 4380ggccaaggaa agctgcagaa ccgcggcact ggtaaacggt aattaagcta tcagccccat 4440gctaacgagt ttaaattacg tgtattgctg ataaacacca acagagcttt actgaaagat 4500gggagtcacg gtgtggcttc cccactgcga ttattgcaca agcagcgagg gcgaacttga 4560ctgtcgtcgc tgagcagcct gcagtcaaac atacatatat atcaaccgcg aagacgtctg 4620gccttgtaga acacgacgct ccctagcaac acctgccgtg tcagcctcta cggttgttac 4680ttgcattcag gatgctctcc agcgggcgag ctattcaaaa tattcaaagc aggtatctcg 4740tattgccagg attcagctga agcaacaggt gccaaggaaa tctgcgtcgg ttctcatctg 4800ggcttgctcg gtcctggcgt agatctagag tcgacctgca ggcatgcggc gtaatcatgg 4860tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 4920ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 4980ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 5040ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 5100gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 5160atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 5220caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 5280cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 5340taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 5400ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 5460tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 5520gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 5580ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 5640aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 5700agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 5760agctcttgat ccgacaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 5820cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 5880gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 5940atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 6000gagtaaactt ggtctgacag ttaagcaagg attttcttaa cttcttcggc gacagcatca 6060ccgacttcgg tggtactgtt ggaaccacct aaatcaccag ttctgatacc tgcatccaaa 6120acctttttaa ctgcatcttc aatggcctta ccttcttcag gcaagttcaa tgacaatttc 6180aacatcattg cagcagacaa gatagtggcg atagggttga ccttattctt tggcaaatct 6240ggagcagaac cgtggcatgg ttcgtacaaa ccaaatgcgg tgttcttgtc tggcaaagag 6300gccaaggacg cagatggcaa caaacccaag gaacctggga taacggaggc ttcatcggag 6360atgatatcac caaacatgtt gctggtgatt ataataccat ttaggtgggt tgggttctta 6420actaggatca tggcggcaga atcaatcaat tgatgttgaa ccttcaatgt agggaattcg 6480ttcttgatgg tttcctccac agtttttctc cataatcttg aagaggccaa aacattagct 6540ttatccaagg accaaatagg caatggtggc tcatgttgta gggccatgaa agcggccatt 6600cttgtgattc tttgcacttc tggaacggtg tattgttcac tatcccaagc gacaccatca 6660ccatcgtctt cctttctctt accaaagtaa atacctccca ctaattctct gacaacaacg 6720aagtcagtac ctttagcaaa ttgtggcttg attggagata agtctaaaag agagtcggat 6780gcaaagttac atggtcttaa gttggcgtac aattgaagtt ctttacggat ttttagtaaa 6840ccttgttcag gtctaacact gccggtaccc catttaggac cacccacagc acctaacaaa 6900acggcatcag ccttcttgga ggcttccagc gcctcatctg gaagtggaac acctgtagca 6960tcgatagcag caccaccaat taaatgattt tcgaaatcga acttgacatt ggaacgaaca 7020tcagaaatag ctttaagaac cttaatggct tcggctgtga tttcttgacc aacgtggtca 7080cctggcaaaa cgacgatctt cttaggggca gacatactct tcctttttca atattattga 7140agcatttatc agggttattg tctcatgagc ggatacatat ttgaa 7185238009DNAArtificial Sequencecomplete DNA sequence of plasmid pMT3328 23tgtataagct agcttatggt gttttgatca ttttaaattt ttatatggcg ggtggtgggc 60aactcgcttg cgcgggcaac tcgcttaccg attacgttag ggctgatatt tacgtaaaaa 120tcgtcaaggg atgcaagacc aaaccgttaa atttccggag tcaacagcat ccaagcccaa 180gtccttcacg gagaaacccc agcgtccaca tcacgagcga aggaccacct ctaggcatcg 240gacgcaccat ccaattagaa gcagcaaagc gaaacagccc aagaaaaagg tcggcccgtc 300ggccttttct gcaacgctga tcacgggcag cgatccaacc aacaccctcc agagtgacta 360ggggcggaaa tttatcggga ttaatttcca ctcaaccaca aatcacagtc gtccccggta 420atttaacggc tgcagacggc aatttaacgg cttctgcgaa tcgcttggat tccccgcccc 480tggccgtaga gcttaaagta tgtcccttgt cgatgcgatg tatgaattca tggtgttttg 540atcattttaa atttttatat ggcgggtggt gggcaactcg cttgcgcggg caactcgctt 600accgattacg ttagggctga tatttacgta aaaatcgtca agggatgcaa gaccaaaccg 660ttaaatttcc ggagtcaaca gcatccaagc ccaagtcctt cacggagaaa ccccagcgtc 720cacatcacga gcgaaggacc acctctaggc atcggacgca ccatccaatt agaagcagca 780aagcgaaaca gcccaagaaa aaggtcggcc cgtcggcctt ttctgcaacg ctgatcacgg 840gcagcgatcc aaccaacacc ctccagagtg actaggggcg gaaatttatc gggattaatt 900tccactcaac cacaaatcac agtcgtcccc ggtaatttaa cggctgcaga cggcaattta 960acggcttctg cgaatcgctt ggattccccg cccctggccg tagagcttaa agtatgtccc 1020ttgtcgatgc gatgtatcac aacatataaa tactggcaag ggatgccatg cttggagttt 1080ccaactcaat ttacctctat ccacacttct cttccttcct caatcctcta tatacacaac 1140tggggatccg tcgaccttgg ggaagtcatc accgaatcta attacgatgt cctgggagct 1200caatatatgg tgaagcagtg ccggaagtcg gtgaacttcc ttggtgctct tgaggccacc 1260agatatgcga aattgatgac tgataagact gtctggctgg aagttggtgc ccataccatt 1320tgctctggta tgatcaaagc aacattcggt ccccaggtta ccactgtggc atctcttcgc 1380cgagaggaga atgcatggaa ggtcctctcc aatagtctat cggcccttca tttggctggc 1440attgatatta attggaaaga atatcatcaa gacttcagct ccagccacca ggtgctccca 1500cttccttctt acaagtggga tctcaagaac tactggatac cctacactaa caatttctgc 1560cttacgaagg gtgctcccca aactgcaatt caagctgcac cacaaactac attcctgacc 1620actgctgcgc aaaaggttgt tgagagtcgc gacgagaatt caatgattaa tgaccttttc 1680ctaaatatac taatactgca cgaccatgct ttcttatcta tttcgggggt tactactgaa 1740atgcaatgag tgactagtag ccagtgacat gtacatgaag tatccttcct acggtctata 1800ggcaaccatt gagatccggt gtctcagacc catatcatag accgttcatt ggaccatgtt 1860atctttgaaa gaatacctaa ggaaaccacc tcggcagata atcacccaag ccccaatagt 1920tctcgcgacc catgggatgt ccggcacact tccgtatctc atgcacctcc acaccctcgg 1980ggagattatc aggatcgtaa tacatgactc caagacgtcc gcaaagttct tcgctagccc 2040ccgggaccgg gaccagagca gcccgggcga ctgcggcaga tccgacgatc agtagaaggt 2100tgagggagag tctcatggtg ggatatggtc ttcctctatt agaggtgagg ttaatatttt 2160gcaggtagag tggataggac gtcggacctg acgtacttga ctttgggata cctaggctcg 2220agatctagag ggtgactgac acctggcggt agacaatcaa tccatttcgc tatagttaaa 2280ggatggggat gagggcaatt ggttatatga tcatgtatgt agtgggtgtg cataatagta 2340gtgaaatgga agccaagtca tgtgattgta atcgaccgac ggaattgagg atatccggaa 2400atacagacac cgtgaaagcc atggtctttc cttcgtgtag aagaccagac agacagtccc 2460tgatttaccc ttgcacaaag cactagaaaa ttagcattcc atccttctct gcttgctctg 2520ctgatatcac tgtcattcaa tgcatagcca tgagctcatc ttagatccaa gcacgtaatt 2580ccatagccga ggtccacagt ggagcagcaa cattccccat cattgctttc cccaggggcc 2640tcccaacgac taaatcaaga gtatatctct accgtccaat agatcgtctt cgcttcaaaa 2700tctttgacaa ttccaagagg gtccccatcc atcaaaccca gttcaataat agccgagatg 2760catggtggag tcaattaggc agtattgctg gaatgtcggg gccagttggc cgggtggtca 2820ttggccgcct gtgatgccat ctgccactaa atccgatcat tgatccaccg cccacgaggc 2880gcgtctttgc tttttgcgcg gcgtccaggt tcaactctct cctctagact ggaaacgcaa 2940ccctgaaggg attcttcctt tgagagatgg aagcgtgtca tatctcttcg gttctacggc 3000aggttttttt ctgctctttc gtagcatggc atggtcactt cagcgcttat ttacagttgc 3060tggtattgat ttcttgtgca aattgctatc tgacacttat tagctatgga gtcaccacat 3120ttcccagcaa cttccccact tcctctgcaa tcgccaacgt cctctcttca ctgagtctcc 3180gtccgataac ctgcactgca accggtgccc catggtacgc ctccggatca tactcttcct 3240gcacgagggc atcaagctca ctaaccgcct tgaaactctc attcttctta tcgatgttct 3300tatccgcaaa ggtaaccgga acaaccacgc tcgtgaaatc cagcaggttg atcacagagg 3360catacccata gtaccggaac tggtcatgcc gtaccgcagc ggtaggcgta atcggcgcga 3420tgatggcgtc cagttccttc ccggcctttt cttcagcctc ccgccatttc tcaaggtact 3480ccatctggta attccacttc tggagatgcg tgtcccagag ctcgttcatg ttaacagctt 3540tgatgttcgg gttcagtagg tctttgatat ttggaatcgc cggctcgccg gatgcactga 3600tatcgcgcat tacgtcggcg ctgccgtcag ccgcgtagat atgggagatg agatcgtggc 3660cgaaatcgtg cttgtatggc gtccacgggg tcacggtgtg accggctttg gcgagtgcgg 3720cgacggtggt ttccacgccg cgcaggatag gagggtgtgg aaggacattg ccgtcgaagt 3780tgtagtagcc gatattgagc ccgccgttct tgatcttgga ggcaataatg tccgactcgg 3840actggcgcca gggcatgggg atgaccttgg agtcgtattt ccatggctcc tgaccgagga 3900cggatttggt gaagaggcgg aggtctaaca tacttcatca gtgactgccg gtctcgtata 3960tagtataaaa agcaagaaag gaggacagtg gaggcctggt atagagcagg aaaagaagga 4020agaggcgaag gactcaccct caacagagtg cgtaatcggc ccgacaacgc tgtgcaccgt 4080ctcctgaccc tccatgctgt tcgccatctt tgcatacggc agccgcccat gactcggcct 4140tagaccgtac aggaagttga acgcggccgg cactcgaatc gagccaccga tatccgttcc 4200tacaccgatg acgccaccac gaatcccaac gatcgcaccc tcaccaccag aactgccgcc 4260gcacgaccag ttcttgttgc gtgggttgac ggtgcgcccg atgatgttgt tgactgtctc 4320gcagaccatc agggtctgcg ggacagaggt cttgacgtag aagacggcac cggctttgcg 4380gagcatggtt gtcagaaccg agtccccttc gtcgtacttg tttagccatg agatgtagcc 4440cattgatgtt tcgtagccct ggtggcatat gttagctgac aaaaagggac atctaacgac 4500ttaggggcaa cggtgtacct tgactcgaag ctggtctttg agagagatgg ggaggccatg 4560gagtggacca acgggtctct tgtgctttgc gtagtattca tcgagttccc ttgcctgcgc 4620gagagcggcg tcagggaaga actcgtgggc gcagtttgtc tgcacagaag ccagcgtcag 4680cttgatagtc ccataaggtg gcgttgttac atctccctga gaggtagagg ggaccctact 4740aactgctggg cgattgctgc ccgtttacag aatgctagcg taacttccac cgaggtcaac 4800tctccggccg ccagcttgga cacaagatct gcagcggagg cctctgtgat cttcagttcg 4860gcctctgaaa ggatcaccga tttctttggg aaatcaataa cgctgtcttc cgcaggcagc 4920gtctggactt tccattcatc agggatggtt tttgcgaggc gggcgcgctt atcagcggcc 4980agttcttccc aggattgagg cattctgtgt tagcttatag tcaggatgtt ggctcgacga 5040gtgtaaactg ggagttggca

tgagggttat gtaggcttct ttagccccgc atccccctca 5100ttctcctcat tgatcccggg ggagcggatg gtgttgataa gagactaatt atagggttta 5160gctggtgcct agctggtgat tggctggctt cgccgaattt tacgggccaa ggaaagctgc 5220agaaccgcgg cactggtaaa cggtaattaa gctatcagcc ccatgctaac gagtttaaat 5280tacgtgtatt gctgataaac accaacagag ctttactgaa agatgggagt cacggtgtgg 5340cttccccact gcgattattg cacaagcagc gagggcgaac ttgactgtcg tcgctgagca 5400gcctgcagtc aaacatacat atatatcaac cgcgaagacg tctggccttg tagaacacga 5460cgctccctag caacacctgc cgtgtcagcc tctacggttg ttacttgcat tcaggatgct 5520ctccagcggg cgagctattc aaaatattca aagcaggtat ctcgtattgc caggattcag 5580ctgaagcaac aggtgccaag gaaatctgcg tcggttctca tctgggcttg ctcggtcctg 5640gcgtagatct agagtcgacc tgcaggcatg cggcgtaatc atggtcatag ctgtttcctg 5700tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 5760aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 5820ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 5880gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 5940tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 6000aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 6060gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 6120aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 6180ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 6240tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 6300tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 6360ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 6420tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 6480ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 6540tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccgaca 6600aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 6660aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 6720aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 6780ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 6840acagttaagc aaggattttc ttaacttctt cggcgacagc atcaccgact tcggtggtac 6900tgttggaacc acctaaatca ccagttctga tacctgcatc caaaaccttt ttaactgcat 6960cttcaatggc cttaccttct tcaggcaagt tcaatgacaa tttcaacatc attgcagcag 7020acaagatagt ggcgataggg ttgaccttat tctttggcaa atctggagca gaaccgtggc 7080atggttcgta caaaccaaat gcggtgttct tgtctggcaa agaggccaag gacgcagatg 7140gcaacaaacc caaggaacct gggataacgg aggcttcatc ggagatgata tcaccaaaca 7200tgttgctggt gattataata ccatttaggt gggttgggtt cttaactagg atcatggcgg 7260cagaatcaat caattgatgt tgaaccttca atgtagggaa ttcgttcttg atggtttcct 7320ccacagtttt tctccataat cttgaagagg ccaaaacatt agctttatcc aaggaccaaa 7380taggcaatgg tggctcatgt tgtagggcca tgaaagcggc cattcttgtg attctttgca 7440cttctggaac ggtgtattgt tcactatccc aagcgacacc atcaccatcg tcttcctttc 7500tcttaccaaa gtaaatacct cccactaatt ctctgacaac aacgaagtca gtacctttag 7560caaattgtgg cttgattgga gataagtcta aaagagagtc ggatgcaaag ttacatggtc 7620ttaagttggc gtacaattga agttctttac ggatttttag taaaccttgt tcaggtctaa 7680cactgccggt accccattta ggaccaccca cagcacctaa caaaacggca tcagccttct 7740tggaggcttc cagcgcctca tctggaagtg gaacacctgt agcatcgata gcagcaccac 7800caattaaatg attttcgaaa tcgaacttga cattggaacg aacatcagaa atagctttaa 7860gaaccttaat ggcttcggct gtgatttctt gaccaacgtg gtcacctggc aaaacgacga 7920tcttcttagg ggcagacata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 7980attgtctcat gagcggatac atatttgaa 8009247909DNAArtificial Sequencecomplet DNA sequence of plasmid pMT3330 24tgtataagct agcttatggt gttttgatca ttttaaattt ttatatggcg ggtggtgggc 60aactcgcttg cgcgggcaac tcgcttaccg attacgttag ggctgatatt tacgtaaaaa 120tcgtcaaggg atgcaagacc aaaccgttaa atttccggag tcaacagcat ccaagcccaa 180gtccttcacg gagaaacccc agcgtccaca tcacgagcga aggaccacct ctaggcatcg 240gacgcaccat ccaattagaa gcagcaaagc gaaacagccc aagaaaaagg tcggcccgtc 300ggccttttct gcaacgctga tcacgggcag cgatccaacc aacaccctcc agagtgacta 360ggggcggaaa tttatcggga ttaatttcca ctcaaccaca aatcacagtc gtccccggta 420atttaacggc tgcagacggc aatttaacgg cttctgcgaa tcgcttggat tccccgcccc 480tggccgtaga gcttaaagta tgtcccttgt cgatgcgatg tatgaattca tggtgttttg 540atcattttaa atttttatat ggcgggtggt gggcaactcg cttgcgcggg caactcgctt 600accgattacg ttagggctga tatttacgta aaaatcgtca agggatgcaa gaccaaaccg 660ttaaatttcc ggagtcaaca gcatccaagc ccaagtcctt cacggagaaa ccccagcgtc 720cacatcacga gcgaaggacc acctctaggc atcggacgca ccatccaatt agaagcagca 780aagcgaaaca gcccaagaaa aaggtcggcc cgtcggcctt ttctgcaacg ctgatcacgg 840gcagcgatcc aaccaacacc ctccagagtg actaggggcg gaaatttatc gggattaatt 900tccactcaac cacaaatcac agtcgtcccc ggtaatttaa cggctgcaga cggcaattta 960acggcttctg cgaatcgctt ggattccccg cccctggccg tagagcttaa agtatgtccc 1020ttgtcgatgc gatgtatcac aacatataaa tactggcaag ggatgccatg cttggagttt 1080ccaactcaat ttacctctat ccacacttct cttccttcct caatcctcta tatacacaac 1140tggggatccg tcgaccttgg ggaagtcatc accgaatcta attacgatgt cctgggagct 1200caatatatgg tgaagcagtg ccggaagtcg gtgaacttcc ttggtgctct tgaggccacc 1260agatatgcga aattgatgac tgataagact gtctggctgg aagttggtgc ccataccatt 1320tgctctggta tgatcaaagc aacattcggt ccccaggtta ccactgtggc atctcttcgc 1380cgagaggaga atgcatggaa ggtcctctcc aatagtctat cggcccttca tttggctggc 1440attgatatta attggaaaga atatcatcaa gacttcagct ccagccacca ggtgctccca 1500cttccttctt acaagtggga tctcaagaac tactggatac cctacactaa caatttctgc 1560cttacgaagg gtgctcccca aactgcaatt caagctgcac cacaaactac attcctgacc 1620actgctgcgc aaaaggttgt tgagagtcgc gacgagaatt caatgattaa tgaccttttc 1680ctaaatatac taatactgca cgaccatgct ttcttatcta tttcgggggt tactactgaa 1740atgcaatgag tgactagtag ccagtgacat gtacatgaag tatccttcct acggtctata 1800ggcaaccatt gagatccggt gtctcagacc catatcatag accgttcatt ggaccatgtt 1860atctttgaaa gaatacctaa ggaaaccacc tcggcagata atcacccaag ccccaatagt 1920tctcgcgacc catgggatgt ccggcacact tccgtatctc atgcacctcc acaccctcgg 1980ggagattatc aggatcgtaa tacatgactc caagacgtcc gcaaagttct tcgctagccc 2040ccgggaccgg gaccagagca gcccgggcga ctgcggcaga tccgacgatc agtagaaggt 2100tgagggagag cctaggctcg agatctagag ggtgactgac acctggcggt agacaatcaa 2160tccatttcgc tatagttaaa ggatggggat gagggcaatt ggttatatga tcatgtatgt 2220agtgggtgtg cataatagta gtgaaatgga agccaagtca tgtgattgta atcgaccgac 2280ggaattgagg atatccggaa atacagacac cgtgaaagcc atggtctttc cttcgtgtag 2340aagaccagac agacagtccc tgatttaccc ttgcacaaag cactagaaaa ttagcattcc 2400atccttctct gcttgctctg ctgatatcac tgtcattcaa tgcatagcca tgagctcatc 2460ttagatccaa gcacgtaatt ccatagccga ggtccacagt ggagcagcaa cattccccat 2520cattgctttc cccaggggcc tcccaacgac taaatcaaga gtatatctct accgtccaat 2580agatcgtctt cgcttcaaaa tctttgacaa ttccaagagg gtccccatcc atcaaaccca 2640gttcaataat agccgagatg catggtggag tcaattaggc agtattgctg gaatgtcggg 2700gccagttggc cgggtggtca ttggccgcct gtgatgccat ctgccactaa atccgatcat 2760tgatccaccg cccacgaggc gcgtctttgc tttttgcgcg gcgtccaggt tcaactctct 2820cctctagact ggaaacgcaa ccctgaaggg attcttcctt tgagagatgg aagcgtgtca 2880tatctcttcg gttctacggc aggttttttt ctgctctttc gtagcatggc atggtcactt 2940cagcgcttat ttacagttgc tggtattgat ttcttgtgca aattgctatc tgacacttat 3000tagctatgga gtcaccacat ttcccagcaa cttccccact tcctctgcaa tcgccaacgt 3060cctctcttca ctgagtctcc gtccgataac ctgcactgca accggtgccc catggtacgc 3120ctccggatca tactcttcct gcacgagggc atcaagctca ctaaccgcct tgaaactctc 3180attcttctta tcgatgttct tatccgcaaa ggtaaccgga acaaccacgc tcgtgaaatc 3240cagcaggttg atcacagagg catacccata gtaccggaac tggtcatgcc gtaccgcagc 3300ggtaggcgta atcggcgcga tgatggcgtc cagttccttc ccggcctttt cttcagcctc 3360ccgccatttc tcaaggtact ccatctggta attccacttc tggagatgcg tgtcccagag 3420ctcgttcatg ttaacagctt tgatgttcgg gttcagtagg tctttgatat ttggaatcgc 3480cggctcgccg gatgcactga tatcgcgcat tacgtcggcg ctgccgtcag ccgcgtagat 3540atgggagatg agatcgtggc cgaaatcgtg cttgtatggc gtccacgggg tcacggtgtg 3600accggctttg gcgagtgcgg cgacggtggt ttccacgccg cgcaggatag gagggtgtgg 3660aaggacattg ccgtcgaagt tgtagtagcc gatattgagc ccgccgttct tgatcttgga 3720ggcaataatg tccgactcgg actggcgcca gggcatgggg atgaccttgg agtcgtattt 3780ccatggctcc tgaccgagga cggatttggt gaagaggcgg aggtctaaca tacttcatca 3840gtgactgccg gtctcgtata tagtataaaa agcaagaaag gaggacagtg gaggcctggt 3900atagagcagg aaaagaagga agaggcgaag gactcaccct caacagagtg cgtaatcggc 3960ccgacaacgc tgtgcaccgt ctcctgaccc tccatgctgt tcgccatctt tgcatacggc 4020agccgcccat gactcggcct tagaccgtac aggaagttga acgcggccgg cactcgaatc 4080gagccaccga tatccgttcc tacaccgatg acgccaccac gaatcccaac gatcgcaccc 4140tcaccaccag aactgccgcc gcacgaccag ttcttgttgc gtgggttgac ggtgcgcccg 4200atgatgttgt tgactgtctc gcagaccatc agggtctgcg ggacagaggt cttgacgtag 4260aagacggcac cggctttgcg gagcatggtt gtcagaaccg agtccccttc gtcgtacttg 4320tttagccatg agatgtagcc cattgatgtt tcgtagccct ggtggcatat gttagctgac 4380aaaaagggac atctaacgac ttaggggcaa cggtgtacct tgactcgaag ctggtctttg 4440agagagatgg ggaggccatg gagtggacca acgggtctct tgtgctttgc gtagtattca 4500tcgagttccc ttgcctgcgc gagagcggcg tcagggaaga actcgtgggc gcagtttgtc 4560tgcacagaag ccagcgtcag cttgatagtc ccataaggtg gcgttgttac atctccctga 4620gaggtagagg ggaccctact aactgctggg cgattgctgc ccgtttacag aatgctagcg 4680taacttccac cgaggtcaac tctccggccg ccagcttgga cacaagatct gcagcggagg 4740cctctgtgat cttcagttcg gcctctgaaa ggatcaccga tttctttggg aaatcaataa 4800cgctgtcttc cgcaggcagc gtctggactt tccattcatc agggatggtt tttgcgaggc 4860gggcgcgctt atcagcggcc agttcttccc aggattgagg cattctgtgt tagcttatag 4920tcaggatgtt ggctcgacga gtgtaaactg ggagttggca tgagggttat gtaggcttct 4980ttagccccgc atccccctca ttctcctcat tgatcccggg ggagcggatg gtgttgataa 5040gagactaatt atagggttta gctggtgcct agctggtgat tggctggctt cgccgaattt 5100tacgggccaa ggaaagctgc agaaccgcgg cactggtaaa cggtaattaa gctatcagcc 5160ccatgctaac gagtttaaat tacgtgtatt gctgataaac accaacagag ctttactgaa 5220agatgggagt cacggtgtgg cttccccact gcgattattg cacaagcagc gagggcgaac 5280ttgactgtcg tcgctgagca gcctgcagtc aaacatacat atatatcaac cgcgaagacg 5340tctggccttg tagaacacga cgctccctag caacacctgc cgtgtcagcc tctacggttg 5400ttacttgcat tcaggatgct ctccagcggg cgagctattc aaaatattca aagcaggtat 5460ctcgtattgc caggattcag ctgaagcaac aggtgccaag gaaatctgcg tcggttctca 5520tctgggcttg ctcggtcctg gcgtagatct agagtcgacc tgcaggcatg cggcgtaatc 5580atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 5640agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 5700tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 5760aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 5820cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 5880ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 5940ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 6000cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 6060actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 6120cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 6180tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 6240gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 6300caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 6360agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 6420tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 6480tggtagctct tgatccgaca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 6540gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 6600gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 6660aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 6720atatgagtaa acttggtctg acagttaagc aaggattttc ttaacttctt cggcgacagc 6780atcaccgact tcggtggtac tgttggaacc acctaaatca ccagttctga tacctgcatc 6840caaaaccttt ttaactgcat cttcaatggc cttaccttct tcaggcaagt tcaatgacaa 6900tttcaacatc attgcagcag acaagatagt ggcgataggg ttgaccttat tctttggcaa 6960atctggagca gaaccgtggc atggttcgta caaaccaaat gcggtgttct tgtctggcaa 7020agaggccaag gacgcagatg gcaacaaacc caaggaacct gggataacgg aggcttcatc 7080ggagatgata tcaccaaaca tgttgctggt gattataata ccatttaggt gggttgggtt 7140cttaactagg atcatggcgg cagaatcaat caattgatgt tgaaccttca atgtagggaa 7200ttcgttcttg atggtttcct ccacagtttt tctccataat cttgaagagg ccaaaacatt 7260agctttatcc aaggaccaaa taggcaatgg tggctcatgt tgtagggcca tgaaagcggc 7320cattcttgtg attctttgca cttctggaac ggtgtattgt tcactatccc aagcgacacc 7380atcaccatcg tcttcctttc tcttaccaaa gtaaatacct cccactaatt ctctgacaac 7440aacgaagtca gtacctttag caaattgtgg cttgattgga gataagtcta aaagagagtc 7500ggatgcaaag ttacatggtc ttaagttggc gtacaattga agttctttac ggatttttag 7560taaaccttgt tcaggtctaa cactgccggt accccattta ggaccaccca cagcacctaa 7620caaaacggca tcagccttct tggaggcttc cagcgcctca tctggaagtg gaacacctgt 7680agcatcgata gcagcaccac caattaaatg attttcgaaa tcgaacttga cattggaacg 7740aacatcagaa atagctttaa gaaccttaat ggcttcggct gtgatttctt gaccaacgtg 7800gtcacctggc aaaacgacga tcttcttagg ggcagacata ctcttccttt ttcaatatta 7860ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaa 7909258964DNAArtificial Sequencecomplete DNA sequence of plasmid pMT3332 25tgtataagct agcttatggt gttttgatca ttttaaattt ttatatggcg ggtggtgggc 60aactcgcttg cgcgggcaac tcgcttaccg attacgttag ggctgatatt tacgtaaaaa 120tcgtcaaggg atgcaagacc aaaccgttaa atttccggag tcaacagcat ccaagcccaa 180gtccttcacg gagaaacccc agcgtccaca tcacgagcga aggaccacct ctaggcatcg 240gacgcaccat ccaattagaa gcagcaaagc gaaacagccc aagaaaaagg tcggcccgtc 300ggccttttct gcaacgctga tcacgggcag cgatccaacc aacaccctcc agagtgacta 360ggggcggaaa tttatcggga ttaatttcca ctcaaccaca aatcacagtc gtccccggta 420atttaacggc tgcagacggc aatttaacgg cttctgcgaa tcgcttggat tccccgcccc 480tggccgtaga gcttaaagta tgtcccttgt cgatgcgatg tatgaattca tggtgttttg 540atcattttaa atttttatat ggcgggtggt gggcaactcg cttgcgcggg caactcgctt 600accgattacg ttagggctga tatttacgta aaaatcgtca agggatgcaa gaccaaaccg 660ttaaatttcc ggagtcaaca gcatccaagc ccaagtcctt cacggagaaa ccccagcgtc 720cacatcacga gcgaaggacc acctctaggc atcggacgca ccatccaatt agaagcagca 780aagcgaaaca gcccaagaaa aaggtcggcc cgtcggcctt ttctgcaacg ctgatcacgg 840gcagcgatcc aaccaacacc ctccagagtg actaggggcg gaaatttatc gggattaatt 900tccactcaac cacaaatcac agtcgtcccc ggtaatttaa cggctgcaga cggcaattta 960acggcttctg cgaatcgctt ggattccccg cccctggccg tagagcttaa agtatgtccc 1020ttgtcgatgc gatgtatcac aacatataaa tactggcaag ggatgccatg cttggagttt 1080ccaactcaat ttacctctat ccacacttct cttccttcct caatcctcta tatacacaac 1140tggggatccg tcgaccttgg ggaagtcatc accgaatcta attacgatgt cctgggagct 1200caatatatgg tgaagcagtg ccggaagtcg gtgaacttcc ttggtgctct tgaggccacc 1260agatatgcga aattgatgac tgataagact gtctggctgg aagttggtgc ccataccatt 1320tgctctggta tgatcaaagc aacattcggt ccccaggtta ccactgtggc atctcttcgc 1380cgagaggaga atgcatggaa ggtcctctcc aatagtctat cggcccttca tttggctggc 1440attgatatta attggaaaga atatcatcaa gacttcagct ccagccacca ggtgctccca 1500cttccttctt acaagtggga tctcaagaac tactggatac cctacactaa caatttctgc 1560cttacgaagg gtgctcccca aactgcaatt caagctgcac cacaaactac attcctgacc 1620actgctgcgc aaaaggttgt tgagagtcgc gacgagaatt caatgattaa tgaccttttc 1680ctaaatatac taatactgca cgaccatgct ttcttatcta tttcgggggt tactactgaa 1740atgcaatgag tgactagtag ccagtgacat gtacatgaag tatccttcct acggtctata 1800ggcaaccatt gagatccggt gtctcagacc catatcatag accgttcatt ggaccatgtt 1860atctttgaaa gaatacctaa ggaaaccacc tcggcagata atcacccaag ccccaatagt 1920tctcgcgacc catgggatgt ccggcacact tccgtatctc atgcacctcc acaccctcgg 1980ggagattatc aggatcgtaa tacatgactc caagacgtcc gcaaagttct tcgctagccc 2040ccgggaccgg gaccagagca gcccgggcga ctgcggcaga tccgacgatc agtagaaggt 2100tgagggagag tctcatggtg ggatatggtc ttcctctatt agaggtgagg ttaatatttt 2160gcaggtagag tggataggac gtcggacctg acgtacttga ctttgggata cctaggctct 2220ccctcaacct tctactgatc gtcggatctg ccgcagtcgc ccgggctgct ctggtcccgg 2280tcccgggggc tagcgaagaa ctttgcggac gtcttggagt catgtattac gatcctgata 2340atctccccga gggtgtggag gtgcatgaga tacggaagtg tgccggacat cccatgggtc 2400gcgagaacta ttggggcttg ggtgattatc tgccgaggtg gtttccttag gtattctttc 2460aaagataaca tggtccaatg aacggtctat gatatgggtc tgagacaccg gatctcaatg 2520gttgcctata gaccgtagga aggatacttc atgtacatgt cactggctac tagtcactca 2580ttgcatttca gtagtaaccc ccgaaataga taagaaagca tggtcgtgca gtattagtat 2640atttaggaaa aggtcattaa tcattgaatt ctcgtcgcga ctctcaacaa ccttttgcgc 2700agcagtggtc aggaatgtag tttgtggtgc agcttgaatt gcagtttggg gagcaccctt 2760cgtaaggcag aaattgttag tgtagggtat ccagtagttc ttgagatccc acttgtaaga 2820aggaagtggg agcacctggt ggctggagct gaagtcttga tgatattctt tccaattaat 2880atcaatgcca gccaaatgaa gggccgatag actattggag aggaccttcc atgcattctc 2940ctctcggcga agagatgcca cagtggtaac ctggggaccg aatgttgctt tgatcatacc 3000agagcaaatg gtatgggcac caacttccag ccagacagtc ttatcagtca tcaatttcgc 3060atatctggtg gcctcaagag caccaaggaa gttcaccgac ttccggcact gcttcaccat 3120atattgagct cccaggacat cgtaattaga ttcggtgatg acttccccaa ggtcgagatc 3180tagagggtga ctgacacctg gcggtagaca atcaatccat ttcgctatag ttaaaggatg 3240gggatgaggg caattggtta tatgatcatg tatgtagtgg gtgtgcataa tagtagtgaa 3300atggaagcca agtcatgtga ttgtaatcga ccgacggaat tgaggatatc cggaaataca 3360gacaccgtga aagccatggt ctttccttcg tgtagaagac cagacagaca gtccctgatt 3420tacccttgca caaagcacta gaaaattagc attccatcct tctctgcttg ctctgctgat 3480atcactgtca ttcaatgcat agccatgagc tcatcttaga tccaagcacg taattccata 3540gccgaggtcc acagtggagc agcaacattc cccatcattg ctttccccag gggcctccca 3600acgactaaat caagagtata tctctaccgt ccaatagatc gtcttcgctt caaaatcttt 3660gacaattcca agagggtccc catccatcaa acccagttca ataatagccg agatgcatgg 3720tggagtcaat taggcagtat tgctggaatg tcggggccag ttggccgggt ggtcattggc 3780cgcctgtgat gccatctgcc actaaatccg atcattgatc caccgcccac gaggcgcgtc 3840tttgcttttt gcgcggcgtc caggttcaac tctctcctct agactggaaa cgcaaccctg 3900aagggattct tcctttgaga gatggaagcg tgtcatatct cttcggttct acggcaggtt 3960tttttctgct ctttcgtagc atggcatggt cacttcagcg cttatttaca gttgctggta 4020ttgatttctt gtgcaaattg

ctatctgaca cttattagct atggagtcac cacatttccc 4080agcaacttcc ccacttcctc tgcaatcgcc aacgtcctct cttcactgag tctccgtccg 4140ataacctgca ctgcaaccgg tgccccatgg tacgcctccg gatcatactc ttcctgcacg 4200agggcatcaa gctcactaac cgccttgaaa ctctcattct tcttatcgat gttcttatcc 4260gcaaaggtaa ccggaacaac cacgctcgtg aaatccagca ggttgatcac agaggcatac 4320ccatagtacc ggaactggtc atgccgtacc gcagcggtag gcgtaatcgg cgcgatgatg 4380gcgtccagtt ccttcccggc cttttcttca gcctcccgcc atttctcaag gtactccatc 4440tggtaattcc acttctggag atgcgtgtcc cagagctcgt tcatgttaac agctttgatg 4500ttcgggttca gtaggtcttt gatatttgga atcgccggct cgccggatgc actgatatcg 4560cgcattacgt cggcgctgcc gtcagccgcg tagatatggg agatgagatc gtggccgaaa 4620tcgtgcttgt atggcgtcca cggggtcacg gtgtgaccgg ctttggcgag tgcggcgacg 4680gtggtttcca cgccgcgcag gataggaggg tgtggaagga cattgccgtc gaagttgtag 4740tagccgatat tgagcccgcc gttcttgatc ttggaggcaa taatgtccga ctcggactgg 4800cgccagggca tggggatgac cttggagtcg tatttccatg gctcctgacc gaggacggat 4860ttggtgaaga ggcggaggtc taacatactt catcagtgac tgccggtctc gtatatagta 4920taaaaagcaa gaaaggagga cagtggaggc ctggtataga gcaggaaaag aaggaagagg 4980cgaaggactc accctcaaca gagtgcgtaa tcggcccgac aacgctgtgc accgtctcct 5040gaccctccat gctgttcgcc atctttgcat acggcagccg cccatgactc ggccttagac 5100cgtacaggaa gttgaacgcg gccggcactc gaatcgagcc accgatatcc gttcctacac 5160cgatgacgcc accacgaatc ccaacgatcg caccctcacc accagaactg ccgccgcacg 5220accagttctt gttgcgtggg ttgacggtgc gcccgatgat gttgttgact gtctcgcaga 5280ccatcagggt ctgcgggaca gaggtcttga cgtagaagac ggcaccggct ttgcggagca 5340tggttgtcag aaccgagtcc ccttcgtcgt acttgtttag ccatgagatg tagcccattg 5400atgtttcgta gccctggtgg catatgttag ctgacaaaaa gggacatcta acgacttagg 5460ggcaacggtg taccttgact cgaagctggt ctttgagaga gatggggagg ccatggagtg 5520gaccaacggg tctcttgtgc tttgcgtagt attcatcgag ttcccttgcc tgcgcgagag 5580cggcgtcagg gaagaactcg tgggcgcagt ttgtctgcac agaagccagc gtcagcttga 5640tagtcccata aggtggcgtt gttacatctc cctgagaggt agaggggacc ctactaactg 5700ctgggcgatt gctgcccgtt tacagaatgc tagcgtaact tccaccgagg tcaactctcc 5760ggccgccagc ttggacacaa gatctgcagc ggaggcctct gtgatcttca gttcggcctc 5820tgaaaggatc accgatttct ttgggaaatc aataacgctg tcttccgcag gcagcgtctg 5880gactttccat tcatcaggga tggtttttgc gaggcgggcg cgcttatcag cggccagttc 5940ttcccaggat tgaggcattc tgtgttagct tatagtcagg atgttggctc gacgagtgta 6000aactgggagt tggcatgagg gttatgtagg cttctttagc cccgcatccc cctcattctc 6060ctcattgatc ccgggggagc ggatggtgtt gataagagac taattatagg gtttagctgg 6120tgcctagctg gtgattggct ggcttcgccg aattttacgg gccaaggaaa gctgcagaac 6180cgcggcactg gtaaacggta attaagctat cagccccatg ctaacgagtt taaattacgt 6240gtattgctga taaacaccaa cagagcttta ctgaaagatg ggagtcacgg tgtggcttcc 6300ccactgcgat tattgcacaa gcagcgaggg cgaacttgac tgtcgtcgct gagcagcctg 6360cagtcaaaca tacatatata tcaaccgcga agacgtctgg ccttgtagaa cacgacgctc 6420cctagcaaca cctgccgtgt cagcctctac ggttgttact tgcattcagg atgctctcca 6480gcgggcgagc tattcaaaat attcaaagca ggtatctcgt attgccagga ttcagctgaa 6540gcaacaggtg ccaaggaaat ctgcgtcggt tctcatctgg gcttgctcgg tcctggcgta 6600gatctagagt cgacctgcag gcatgcggcg taatcatggt catagctgtt tcctgtgtga 6660aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 6720tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 6780cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 6840ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 6900cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 6960ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 7020aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 7080cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 7140cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 7200gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 7260tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 7320cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 7380ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 7440gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 7500gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cgacaaacaa 7560accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 7620ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 7680tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 7740aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 7800taagcaagga ttttcttaac ttcttcggcg acagcatcac cgacttcggt ggtactgttg 7860gaaccaccta aatcaccagt tctgatacct gcatccaaaa cctttttaac tgcatcttca 7920atggccttac cttcttcagg caagttcaat gacaatttca acatcattgc agcagacaag 7980atagtggcga tagggttgac cttattcttt ggcaaatctg gagcagaacc gtggcatggt 8040tcgtacaaac caaatgcggt gttcttgtct ggcaaagagg ccaaggacgc agatggcaac 8100aaacccaagg aacctgggat aacggaggct tcatcggaga tgatatcacc aaacatgttg 8160ctggtgatta taataccatt taggtgggtt gggttcttaa ctaggatcat ggcggcagaa 8220tcaatcaatt gatgttgaac cttcaatgta gggaattcgt tcttgatggt ttcctccaca 8280gtttttctcc ataatcttga agaggccaaa acattagctt tatccaagga ccaaataggc 8340aatggtggct catgttgtag ggccatgaaa gcggccattc ttgtgattct ttgcacttct 8400ggaacggtgt attgttcact atcccaagcg acaccatcac catcgtcttc ctttctctta 8460ccaaagtaaa tacctcccac taattctctg acaacaacga agtcagtacc tttagcaaat 8520tgtggcttga ttggagataa gtctaaaaga gagtcggatg caaagttaca tggtcttaag 8580ttggcgtaca attgaagttc tttacggatt tttagtaaac cttgttcagg tctaacactg 8640ccggtacccc atttaggacc acccacagca cctaacaaaa cggcatcagc cttcttggag 8700gcttccagcg cctcatctgg aagtggaaca cctgtagcat cgatagcagc accaccaatt 8760aaatgatttt cgaaatcgaa cttgacattg gaacgaacat cagaaatagc tttaagaacc 8820ttaatggctt cggctgtgat ttcttgacca acgtggtcac ctggcaaaac gacgatcttc 8880ttaggggcag acatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 8940ctcatgagcg gatacatatt tgaa 8964266248DNAArtificial Sequencecomplete DNA sequence of plasmid pMT3369 26agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca 240gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc ccttcgatcg 300acttgaaaac catgtctgac aattggatat acaaccgtag cgtatgtatt tgaactaatg 360ggattgtgga ctgtgtatag ccaattctat ttcgtatgat ggatcccttt tcatttaatc 420taactaaggt ggatataact tgaaggctaa cgattgcgtc tcgggtaagg atcaccatca 480ctatcctacg tgcttaaggc ccaggctggc catcaagcag ctcatagagt tctacagaac 540tcttggtaca tactgaaaga aattcctaat tggctttggc tatccgggga aattttagcg 600agcagaaaat atccctacgc tgaggtggac cgctcggtcg atcggtatat tgaccctctt 660taaatcagcc cccaaatctc cgattatgta tgtcatccca ggactttcta caatcaatct 720acttcgcgtg tcaatctacc gattgtggag ccatgtcaga gagaaacatg gtatcaaata 780ttgaatttgg tgcactacac ctagagtcgg acctcagcaa agccatcatt gcatgggatg 840gtcaaatcga cacgcgaaat ccaaggagag aacccgccat gactagcaga gagccctgcg 900ctcagatgaa tcgcaaagcc cgaggattgg tatcagcgaa agtatcatcc acaattttgt 960ctaccgactt gacatagata gtcccttccg atgaatatat tccacctctc ccaaccactg 1020aggctaatat gcagttatca acaatccacg caaaccagct tgcattagac ccttctgctt 1080ggataacatc cttgtttggc aggatttatc tgataggttc aatatagata ctagacttct 1140ataattattc ggagtgatcc aaagctgatt cctatcttat cacccatgcc gtccgacgta 1200tactgccaaa gattataatc agagatgaaa tgttggaaaa tagagctgag cattctgcat 1260ctggtcgttg tggtctgtaa ccttggtgag gccccgtcta aaggaacgga tgctacacaa 1320aactatggtt tgttagcatc caatatgtcg catctcttcc cactactttg attcaaacca 1380tggtacttgg ccgcatgacg taaaatagac ccgctctgga tcaagattag tctaaagctt 1440gtgatagggg gttaccactc attgcatttc agtagtaacc cccgaaatag ataagaaagc 1500atggtcgtgc agtattagta tatttaggaa aaggtcatta atcattacca acacacttgc 1560gttgagtcat cttactcaac cggcctcctt ccccacacaa catgaaactt cccatagata 1620tgcaccctag gatctctcaa ctcattccgc acagaggcca agaaaactgc tacctccacc 1680ggggaccagc cgagaaccct tgtacaaagc gcaagagcat acgactcaag actcataacc 1740atgacttcgc gatggaaccg accggcttct tttaatcgct tttctttagc ccagggaccc 1800atcgggaact acagaaatta agttaatacc cgatagatta acggagtatt aaaccagagg 1860gagagcgaaa gtagaaaaaa cctaccttgt agatcttttc cttcacatcg gtgaaccctg 1920cgtctatcat atttctctga tggtcggccg ccaccttcat tggccggtta aacctcgagc 1980ttgcctcaat caacagtctg aaccactcgc ccagtgaggg cgccagatcg agtgtgtcat 2040cgtcagagaa atgttccatc tcaaagtcgg atagttccaa ccatccacct ggcttcatgt 2100gtgaatatgc ctggcgcagc agtttcggcc agtcggcgat cgaaccggca aggtcgcatg 2160cacggatgaa gtcgaagtaa tctcgtttga aagtccagtc ggcttcgacg tcgtcgacga 2220agaattcgag gttcggaggg acccaggcgg gttggatggg gctgagatcg gtgccgatta 2280ctcgggcgtt ggggtgtgtg tctgcgaagt cgatggccca gataccttta ttattatttg 2340gtcagccatg agcgagcggg tttcctggaa tagatacggc atgtgccata cctgttccgg 2400tgccaatgtc gaaaacgttc tcaacatcct ctgggatcgg ggcgtgaaag agatctccac 2460tgaaatgcag attgcgaacg tggtgtaata ggtcctcgcg atcttgctct tgttcgtcgt 2520ttggcagcac acactgccct tcgcggtagg agtggtatcg acggccattc tcgtacttgt 2580agtttcaagg gcgaattcgc ggccgctaaa ttcaattcgc cctatagtga gtcgtattac 2640aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 2700aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 2760gatcgccctt cccaacagtt gcgcagccta tacgtacggc agtttaaggt ttacacctat 2820aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccg 2880gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt 2940gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg 3000gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat 3060gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg catgagatta 3120tcaaaaagga tcttcaccta gatccttttc acgtagaaag ccagtccgca gaaacggtgc 3180tgaccccgga tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag 3240agaaagcagg tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg 3300acagcaagcg aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc 3360aaagtaaact ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagctct 3420gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt 3480tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc 3540tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag 3600accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg 3660gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac 3720tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc 3780gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc 3840tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc 3900ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg 3960ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat 4020gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc 4080cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa 4140gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat 4200tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgaattat taacgcttac 4260aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatcagg 4320tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 4380aaatatgtat ccgctcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 4440aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 4500atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 4560ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 4620tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 4680agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 4740taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 4800tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 4860cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 4920ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 4980tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 5040tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 5100cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 5160tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 5220gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 5280tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 5340atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 5400tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 5460aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 5520aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 5580tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc 5640gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 5700cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 5760acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 5820cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 5880cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 5940aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 6000gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 6060atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 6120tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 6180gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 6240agcggaag 6248279787DNAArtificial Sequencecomplete DNA sequence of plasmid pMT3376 27agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca 240gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc ccttcgatcg 300acttgaaaac catgtctgac aattggatat acaaccgtag cgtatgtatt tgaactaatg 360ggattgtgga ctgtgtatag ccaattctat ttcgtatgat ggatcccttt tcatttaatc 420taactaaggt ggatataact tgaaggctaa cgattgcgtc tcgggtaagg atcaccatca 480ctatcctacg tgcttaaggc ccaggctggc catcaagcag ctcatagagt tctacagaac 540tcttggtaca tactgaaaga aattcctaat tggctttggc tatccgggga aattttagcg 600agcagaaaat atccctacgc tgaggtggac cgctcggtcg atcggtatat tgaccctctt 660taaatcagcc cccaaatctc cgattatgta tgtcatccca ggactttcta caatcaatct 720acttcgcgtg tcaatctacc gattgtggag ccatgtcaga gagaaacatg gtatcaaata 780ttgaatttgg tgcactacac ctagagtcgg acctcagcaa agccatcatt gcatgggatg 840gtcaaatcga cacgcgaaat ccaaggagag aacccgccat gactagcaga gagccctgcg 900ctcagatgaa tcgcaaagcc cgaggattgg tatcagcgaa agtatcatcc acaattttgt 960ctaccgactt gacatagata gtcccttccg atgaatatat tccacctctc ccaaccactg 1020aggctaatat gcagttatca acaatccacg caaaccagct tgcattagac ccttctgctt 1080ggataacatc cttgtttggc aggatttatc tgataggttc aatatagata ctagacttct 1140ataattattc ggagtgatcc aaagctgatt cctatcttat cacccatgcc gtccgacgta 1200tactgccaaa gattataatc agagatgaaa tgttggaaaa tagagctgag cattctgcat 1260ctggtcgttg tggtctgtaa ccttggtgag gccccgtcta aaggaacgga tgctacacaa 1320aactatggtt tgttagcatc caatatgtcg catctcttcc cactactttg attcaaacca 1380tggtacttgg ccgcatgacg taaaatagac ccgctctgga tcaagattag tctaaagctt 1440ctctggtact cttcgatctc tctcctcggt tgcattggca aggaggttgg cgacatctga 1500tagtacacac caggttagca agactatacc tatgtctacg gaggattcat gactgtgaaa 1560cttacatcta tctaccggta gactagggtc ccgcaaagca ttgagatcca cttgtggtgg 1620ccctgaggcc tctgtcgagg gccgtctgct ggtcgactga gggggactta gcgccgtata 1680ctgagtcgga actttaggca ccccgggcac cgggggcgca tttgcggagt caagatcagg 1740attgatctgt ggatcctatg gatctcagaa caatatacca gaaaatgcga aggtaagtgc 1800ttctatattg atccttagtg ctttcaaact gtgatgtaga agttgctcgg tagctgatta 1860aatattctag acccaagccg ctgctggaat tgacattatt atggccgata gggttgggct 1920tattgctatg tccctgaaag gatatcaaaa gcaggcaaaa agccaggcat aatccccgcg 1980tggacggtac cctaaggata ggccctaatc ttatctacat gtgactgcat cgatgtgttt 2040ggtcaaaatg aggcatgtgg ctcaccccac aggcggagaa acgtgtggct agtgcatgac 2100agtcccctcc atagattcaa tttaattttt cgcggcaatt gtcgtgcagt ttgtatctac 2160atttcattcc atatatcaag agttagtagt tggacatcct gattattttg tctaattact 2220gaaaactcga agtactaacc tactaataag ccagtttcaa ccactaagtg ctcatttata 2280caatatttgc agaaccccgc gctacccctc catcgccaac atgtcttcca agtcgcaatt 2340gacctacagc gcacgcgcta gcaagcaccc caatgcgctc gtaaagaagc tcttcgaggt 2400tgccgaggcc aagaaaacca atgtcaccgt ttccgccgac gtgacaacca ccaaagagct 2460gctggatttg gctgaccgta tgcgcaccgg ggatgccact tacatgtgat ctagtaatgg 2520ttaatggtgg attatataac aggactcggt ccgtacattg ccgtgatcaa aactcacatc 2580gatatcctct ccgatttcag cgaagaaacc atcaccggtc tgaaggccct tgcagagaag 2640cacaatttcc tcatcttcga agatcgcaag ttcatcgata tcggaaacac agtccaaaag 2700cagtaccatg gcggcactct gcgtatctct gagtgggccc acatcatcaa ctgcagtatt 2760ctgcccggtg agggtatcgt cgaggctctg gcccagactg cttcggccga ggacttcccc 2820tacggctccg agaggggcct tttgatcctt gcggagatga cctccaaggg atctttggct 2880accggtcaat atactacttc ttctgttgac tatgctcgga agtataagaa gtttgtgatg 2940ggattcgtct cgacacgtca ccttggcgag gttcagtctg aagttagctc gccttcggag 3000gaggaagatt ttgtcgtctt cacgacaggt gtcaacctct cctcgaaggg tgacaagctg 3060ggacagcagt accaaactcc tgagtcggct gttggacgcg gtgccgactt tattattgct 3120ggccgtggaa tttatgctgc tcctgatccc gtggaggcgg cgaagcagta ccagaaggag 3180ggatgggatg catacctgaa gcgtgttggt gcgcaataag tagtggtgga tacgtactcc 3240ttttatggca gtatgtcgca agtatgatgc gatttataaa ttcagcactc gaaatgacta 3300ctactatgtg tctacgacag ataccctctc cgtacgaata agacacctgc ctcgatatat 3360ggacaaattc aaaatcaggg tcaagggtca tgtttcaaag tcacaacaat ctccaacata 3420gacgagaatt tgtaccggag tgtctgaagg tgcagctgga gattggtcta ttttcttaga 3480gtggggtatc actaatgtac agtcggtcac tatcgtacaa acaatcacaa ttatatacaa 3540gatttcccac caccccctac tctaacacgg cacaattatc catcgagtca gagcctagcc 3600accatttggt gctctcgtag agaccaaagt ataatcctga tccgacagcg gccataaacg 3660tgttgatagc acaccctcgg

aatagtcctc tcgggccatc tgttcgtaca atctcccgta 3720cggtattgat catccttttc ttctgaggtg cagttgtatc tgcagcatcg agcatgattc 3780gtgtccggac catatccatg ggtgctgtca agacactagc tataccgccc gagaccgcag 3840cacttattgc ggctgtcgct gcagcctctc cgattgtcga atgggcctct ttctttccat 3900actctcttgg tctttctagc accttctctc gatctccgaa tctatattca aaaattcgat 3960accgaaaaga ctcgtacaga ggcatctgaa tcgccgacac tggcaagcta tgcgccacaa 4020gagccgggta tccgctccaa agctgtctag ggttgataaa cttcttgaaa gctagccgtg 4080tcgctttctg ggctacacca cctacccttc ccccagctac aggtgctgat gcgtctggat 4140ggtgtgattg gatcatctgc gcgttgtgtt ttaatgcatc agccggagca aagactccgc 4200aagcagcaag atccgcaacg gaggctgcgc aaaaatcgga aaagagccga gctgagctag 4260actcatgcgt tccaagtttt tgatgtatga cttggagtcc tgactgtgca tactcgtatg 4320tgatgaagaa tgcgcccgct ttgacccgtt attttttgct caatgaatat gagttgggta 4380aggataatgt tgggtatcac caacctgtgg gaaatgaggc agcggttacg ctcgcgatac 4440cttggtaaag gccgcggaat atccctgggt gtctccatat gctggttcct gtgttggttc 4500tcaggaactg cgaatattcg cgagactgaa tgcgggtttt gatcgtatct aagggatatg 4560tgaatagatc tctctggtac tcttcgatct ctctcctcgg ttgcattggc aaggaggttg 4620gcgacatctg atagtacaca ccaggttagc aagactatac ctatgtctac ggaggattca 4680tgactgtgaa acttacatct atctaccggt agactagggt cccgcaaagc attgagatcc 4740acttgtggtg gccctgaggc ctctgtcgag ggccgtctgc tggtcgactg agggggactt 4800agcgccgtat actgagtcgg aactttaggc accccgggca ccgggggcgc atttgcggag 4860tcaagatcag gattgatctg tggatcctat ggatctcaga acaatatacc agaaaatgcg 4920aaggtaagtg cttctatatt gatccttagt gctttcaaac tgtgatgtag aagaagcttg 4980tgataggggg ttaccactca ttgcatttca gtagtaaccc ccgaaataga taagaaagca 5040tggtcgtgca gtattagtat atttaggaaa aggtcattaa tcattaccaa cacacttgcg 5100ttgagtcatc ttactcaacc ggcctccttc cccacacaac atgaaacttc ccatagatat 5160gcaccctagg atctctcaac tcattccgca cagaggccaa gaaaactgct acctccaccg 5220gggaccagcc gagaaccctt gtacaaagcg caagagcata cgactcaaga ctcataacca 5280tgacttcgcg atggaaccga ccggcttctt ttaatcgctt ttctttagcc cagggaccca 5340tcgggaacta cagaaattaa gttaataccc gatagattaa cggagtatta aaccagaggg 5400agagcgaaag tagaaaaaac ctaccttgta gatcttttcc ttcacatcgg tgaaccctgc 5460gtctatcata tttctctgat ggtcggccgc caccttcatt ggccggttaa acctcgagct 5520tgcctcaatc aacagtctga accactcgcc cagtgagggc gccagatcga gtgtgtcatc 5580gtcagagaaa tgttccatct caaagtcgga tagttccaac catccacctg gcttcatgtg 5640tgaatatgcc tggcgcagca gtttcggcca gtcggcgatc gaaccggcaa ggtcgcatgc 5700acggatgaag tcgaagtaat ctcgtttgaa agtccagtcg gcttcgacgt cgtcgacgaa 5760gaattcgagg ttcggaggga cccaggcggg ttggatgggg ctgagatcgg tgccgattac 5820tcgggcgttg gggtgtgtgt ctgcgaagtc gatggcccag atacctttat tattatttgg 5880tcagccatga gcgagcgggt ttcctggaat agatacggca tgtgccatac ctgttccggt 5940gccaatgtcg aaaacgttct caacatcctc tgggatcggg gcgtgaaaga gatctccact 6000gaaatgcaga ttgcgaacgt ggtgtaatag gtcctcgcga tcttgctctt gttcgtcgtt 6060tggcagcaca cactgccctt cgcggtagga gtggtatcga cggccattct cgtacttgta 6120gtttcaaggg cgaattcgcg gccgctaaat tcaattcgcc ctatagtgag tcgtattaca 6180attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 6240atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 6300atcgcccttc ccaacagttg cgcagcctat acgtacggca gtttaaggtt tacacctata 6360aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgccgg 6420ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg 6480aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg 6540ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg 6600acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc atgagattat 6660caaaaaggat cttcacctag atccttttca cgtagaaagc cagtccgcag aaacggtgct 6720gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca agcgcaaaga 6780gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga 6840cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg aagccctgca 6900aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagctctg 6960atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt 7020ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct 7080gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga 7140ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg 7200ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact 7260ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg 7320agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct 7380gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg 7440gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt 7500tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg 7560cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc 7620ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag 7680agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt 7740cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaattatt aacgcttaca 7800atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatcaggt 7860ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 7920aatatgtatc cgctcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 7980aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 8040tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 8100tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 8160gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 8220gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 8280aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 8340gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 8400ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 8460tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 8520atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 8580ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 8640ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 8700ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 8760atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 8820gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 8880tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 8940ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 9000aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 9060aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 9120ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 9180tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 9240ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 9300cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 9360agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 9420gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 9480ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 9540tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 9600tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 9660cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 9720tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 9780gcggaag 97872811649DNAArtificial Sequencecomplete DNA sequence of plasmid pMT3378 28gaatacacgg aattcctcga gtaccattta attctatttg tgtttgatcg agacctaata 60cagcccctac aacgaccatc aaagtcgtat agctaccagt gaggaagtgg actcaaatcg 120acttcagcaa catctcctgg ataaacttta agcctaaact atacagaata agataggtgg 180agagcttata ccgagctccc aaatctgtcc agatcatggt tgaccggtgc ctggatcttc 240ctatagaatc atccttattc gttgacctag ctgattctgg agtgacccag agggtcatga 300cttgagccta aaatccgccg cctccaccat ttgtagaaaa atgtgacgaa ctcgtgagct 360ctgtacagtg accggtgact ctttctggca tgcggagaga cggacggacg cagagagaag 420ggctgagtaa taagccactg gccagacagc tctggcggct ctgaggtgca gtggatgatt 480attaatccgg gaccggccgc ccctccgccc cgaagtggaa aggctggtgt gcccctcgtt 540gaccaagaat ctattgcatc atcggagaat atggagcttc atcgaatcac cggcagtaag 600cgaaggagaa tgtgaagcca ggggtgtata gccgtcggcg aaatagcatg ccattaacct 660aggtacagaa gtccaattgc ttccgatctg gtaaaagatt cacgagatag taccttctcc 720gaagtaggta gagcgagtac ccggcgcgta agctccctaa ttggcccatc cggcatctgt 780agggcgtcca aatatcgtgc ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc 840tcaggagctg gccagcggcg cagaccggga acacaagctg gcagtcgacc catccggtgc 900tctgcactcg acctgctgag gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc 960gcccggtgtg tcggcggggt tgacaaggtc gttgcgtcag tccaacattt gttgccatat 1020tttcctgctc tccccaccag ctgtagatct tggtggcgtg aaactcccgc acctcttcgg 1080ccagcgcctt gtagaagcgc gtatggcttc gtaccccggc catcaacacg cgtctgcgtt 1140cgaccaggct gcgcgttctc gcggccatag caaccgacgt acggcgttgc gccctcgccg 1200gcagcaagaa gccacggaag tccgcccgga gcagaaaatg cccacgctac tgcgggttta 1260tatagacggt ccccacggga tggggaaaac caccaccacg caactgctgg tggccctggg 1320ttcgcgcgac gatatcgtct acgtacccga gccgatgact tactggcggg tgctgggggc 1380ttccgagaca atcgcgaaca tctacaccac acaacaccgc ctcgaccagg gtgagatatc 1440ggccggggac gcggcggtgg taatgacaag cgcccagata acaatgggca tgccttatgc 1500cgtgaccgac gccgttctgg ctcctcatat cgggggggag gctgggagct cacatgcccc 1560gcccccggcc ctcaccctca tcttcgaccg ccatcccatc gccgccctcc tgtgctaccc 1620ggccgcgcgg taccttatgg gcagcatgac cccccaggcc gtgctggcgt tcgtggccct 1680catcccgccg accttgcccg gcaccaacat cgtgcttggg gcccttccgg aggacagaca 1740catcgaccgc ctggccaaac gccagcgccc cggcgagcgg ctggacctgg ctatgctggc 1800tgcgattcgc cgcgtttacg ggctacttgc caatacggtg cggtatctgc agtgcggcgg 1860gtcgtggcgg gaggactggg gacagctttc ggggacggcc gtgccgcccc agggtgccga 1920gccccagagc aacgcgggcc cacgacccca tatcggggac acgttattta ccctgtttcg 1980gggccccgag ttgctggccc ccaacggcga cctgtataac gtgtttgcct gggccttgga 2040cgtcttggcc aaacgcctcc gttccatgca cgtctttatc ctggattacg accaatcgcc 2100cgccggctgc cgggacgccc tgctgcaact tacctccggg atggtccaga cccacgtcac 2160cacccccggc tccataccga cgatatgcga cctggcgcgc acgtttgccc gggagatggg 2220ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atccggatcg 2280atccacttaa cgttactgaa atcatcaaac agcttgacga atctggatat aagatcgttg 2340gtgtcgatgt cagctccgga gttgagacaa atggtgttca ggatctcgat aagatacgtt 2400catttgtcca agcagcaaag agtgccttct agtgatttaa tagctccatg tcaacaagaa 2460taaaacgcgt tttcgggttt acctcttcca gatacagctc atctgcaatg cattaatgca 2520ttgactgcaa cctagtaacg ccttncaggc tccggcgaag agaagaatag cttagcagag 2580ctattttcat tttcgggaga cgagatcaag cagatcaacg gtcgtcaaga gacctacgag 2640actgaggaat ccgctcttgg ctccacgcga ctatatattt gtctctaatt gtactttgac 2700atgctcctct tctttactct gatagcttga ctatgaaaat tccgtcacca gcncctgggt 2760tcgcaaagat aattgcatgt ttcttccttg aactctcaag cctacaggac acacattcat 2820cgtaggtata aacctcgaaa tcanttccta ctaagatggt atacaatagt aaccatgcat 2880ggttgcctag tgaatgctcc gtaacaccca atacgccggc cgaaactttt ttacaactct 2940cctatgagtc gtttacccag aatgcacagg tacacttgtt tagaggtaat ccttctttct 3000agtcctgcag gtttaaacga attcgccctt cgatcgactt gaaaaccatg tctgacaatt 3060ggatatacaa ccgtagcgta tgtatttgaa ctaatgggat tgtggactgt gtatagccaa 3120ttctatttcg tatgatggat cccttttcat ttaatctaac taaggtggat ataacttgaa 3180ggctaacgat tgcgtctcgg gtaaggatca ccatcactat cctacgtgct taaggcccag 3240gctggccatc aagcagctca tagagttcta cagaactctt ggtacatact gaaagaaatt 3300cctaattggc tttggctatc cggggaaatt ttagcgagca gaaaatatcc ctacgctgag 3360gtggaccgct cggtcgatcg gtatattgac cctctttaaa tcagccccca aatctccgat 3420tatgtatgtc atcccaggac tttctacaat caatctactt cgcgtgtcaa tctaccgatt 3480gtggagccat gtcagagaga aacatggtat caaatattga atttggtgca ctacacctag 3540agtcggacct cagcaaagcc atcattgcat gggatggtca aatcgacacg cgaaatccaa 3600ggagagaacc cgccatgact agcagagagc cctgcgctca gatgaatcgc aaagcccgag 3660gattggtatc agcgaaagta tcatccacaa ttttgtctac cgacttgaca tagatagtcc 3720cttccgatga atatattcca cctctcccaa ccactgaggc taatatgcag ttatcaacaa 3780tccacgcaaa ccagcttgca ttagaccctt ctgcttggat aacatccttg tttggcagga 3840tttatctgat aggttcaata tagatactag acttctataa ttattcggag tgatccaaag 3900ctgattccta tcttatcacc catgccgtcc gacgtatact gccaaagatt ataatcagag 3960atgaaatgtt ggaaaataga gctgagcatt ctgcatctgg tcgttgtggt ctgtaacctt 4020ggtgaggccc cgtctaaagg aacggatgct acacaaaact atggtttgtt agcatccaat 4080atgtcgcatc tcttcccact actttgattc aaaccatggt acttggccgc atgacgtaaa 4140atagacccgc tctggatcaa gattagtcta aagcttctct ggtactcttc gatctctctc 4200ctcggttgca ttggcaagga ggttggcgac atctgatagt acacaccagg ttagcaagac 4260tatacctatg tctacggagg attcatgact gtgaaactta catctatcta ccggtagact 4320agggtcccgc aaagcattga gatccacttg tggtggccct gaggcctctg tcgagggccg 4380tctgctggtc gactgagggg gacttagcgc cgtatactga gtcggaactt taggcacccc 4440gggcaccggg ggcgcatttg cggagtcaag atcaggattg atctgtggat cctatggatc 4500tcagaacaat ataccagaaa atgcgaaggt aagtgcttct atattgatcc ttagtgcttt 4560caaactgtga tgtagaagtt gctcggtagc tgattaaata ttctagaccc aagccgctgc 4620tggaattgac attattatgg ccgatagggt tgggcttatt gctatgtccc tgaaaggata 4680tcaaaagcag gcaaaaagcc aggcataatc cccgcgtgga cggtacccta aggataggcc 4740ctaatcttat ctacatgtga ctgcatcgat gtgtttggtc aaaatgaggc atgtggctca 4800ccccacaggc ggagaaacgt gtggctagtg catgacagtc ccctccatag attcaattta 4860atttttcgcg gcaattgtcg tgcagtttgt atctacattt cattccatat atcaagagtt 4920agtagttgga catcctgatt attttgtcta attactgaaa actcgaagta ctaacctact 4980aataagccag tttcaaccac taagtgctca tttatacaat atttgcagaa ccccgcgcta 5040cccctccatc gccaacatgt cttccaagtc gcaattgacc tacagcgcac gcgctagcaa 5100gcaccccaat gcgctcgtaa agaagctctt cgaggttgcc gaggccaaga aaaccaatgt 5160caccgtttcc gccgacgtga caaccaccaa agagctgctg gatttggctg accgtatgcg 5220caccggggat gccacttaca tgtgatctag taatggttaa tggtggatta tataacagga 5280ctcggtccgt acattgccgt gatcaaaact cacatcgata tcctctccga tttcagcgaa 5340gaaaccatca ccggtctgaa ggcccttgca gagaagcaca atttcctcat cttcgaagat 5400cgcaagttca tcgatatcgg aaacacagtc caaaagcagt accatggcgg cactctgcgt 5460atctctgagt gggcccacat catcaactgc agtattctgc ccggtgaggg tatcgtcgag 5520gctctggccc agactgcttc ggccgaggac ttcccctacg gctccgagag gggccttttg 5580atccttgcgg agatgacctc caagggatct ttggctaccg gtcaatatac tacttcttct 5640gttgactatg ctcggaagta taagaagttt gtgatgggat tcgtctcgac acgtcacctt 5700ggcgaggttc agtctgaagt tagctcgcct tcggaggagg aagattttgt cgtcttcacg 5760acaggtgtca acctctcctc gaagggtgac aagctgggac agcagtacca aactcctgag 5820tcggctgttg gacgcggtgc cgactttatt attgctggcc gtggaattta tgctgctcct 5880gatcccgtgg aggcggcgaa gcagtaccag aaggagggat gggatgcata cctgaagcgt 5940gttggtgcgc aataagtagt ggtggatacg tactcctttt atggcagtat gtcgcaagta 6000tgatgcgatt tataaattca gcactcgaaa tgactactac tatgtgtcta cgacagatac 6060cctctccgta cgaataagac acctgcctcg atatatggac aaattcaaaa tcagggtcaa 6120gggtcatgtt tcaaagtcac aacaatctcc aacatagacg agaatttgta ccggagtgtc 6180tgaaggtgca gctggagatt ggtctatttt cttagagtgg ggtatcacta atgtacagtc 6240ggtcactatc gtacaaacaa tcacaattat atacaagatt tcccaccacc ccctactcta 6300acacggcaca attatccatc gagtcagagc ctagccacca tttggtgctc tcgtagagac 6360caaagtataa tcctgatccg acagcggcca taaacgtgtt gatagcacac cctcggaata 6420gtcctctcgg gccatctgtt cgtacaatct cccgtacggt attgatcatc cttttcttct 6480gaggtgcagt tgtatctgca gcatcgagca tgattcgtgt ccggaccata tccatgggtg 6540ctgtcaagac actagctata ccgcccgaga ccgcagcact tattgcggct gtcgctgcag 6600cctctccgat tgtcgaatgg gcctctttct ttccatactc tcttggtctt tctagcacct 6660tctctcgatc tccgaatcta tattcaaaaa ttcgataccg aaaagactcg tacagaggca 6720tctgaatcgc cgacactggc aagctatgcg ccacaagagc cgggtatccg ctccaaagct 6780gtctagggtt gataaacttc ttgaaagcta gccgtgtcgc tttctgggct acaccaccta 6840cccttccccc agctacaggt gctgatgcgt ctggatggtg tgattggatc atctgcgcgt 6900tgtgttttaa tgcatcagcc ggagcaaaga ctccgcaagc agcaagatcc gcaacggagg 6960ctgcgcaaaa atcggaaaag agccgagctg agctagactc atgcgttcca agtttttgat 7020gtatgacttg gagtcctgac tgtgcatact cgtatgtgat gaagaatgcg cccgctttga 7080cccgttattt tttgctcaat gaatatgagt tgggtaagga taatgttggg tatcaccaac 7140ctgtgggaaa tgaggcagcg gttacgctcg cgataccttg gtaaaggccg cggaatatcc 7200ctgggtgtct ccatatgctg gttcctgtgt tggttctcag gaactgcgaa tattcgcgag 7260actgaatgcg ggttttgatc gtatctaagg gatatgtgaa tagatctctc tggtactctt 7320cgatctctct cctcggttgc attggcaagg aggttggcga catctgatag tacacaccag 7380gttagcaaga ctatacctat gtctacggag gattcatgac tgtgaaactt acatctatct 7440accggtagac tagggtcccg caaagcattg agatccactt gtggtggccc tgaggcctct 7500gtcgagggcc gtctgctggt cgactgaggg ggacttagcg ccgtatactg agtcggaact 7560ttaggcaccc cgggcaccgg gggcgcattt gcggagtcaa gatcaggatt gatctgtgga 7620tcctatggat ctcagaacaa tataccagaa aatgcgaagg taagtgcttc tatattgatc 7680cttagtgctt tcaaactgtg atgtagaaga agcttgtgat agggggttac cactcattgc 7740atttcagtag taacccccga aatagataag aaagcatggt cgtgcagtat tagtatattt 7800aggaaaaggt cattaatcat taccaacaca cttgcgttga gtcatcttac tcaaccggcc 7860tccttcccca cacaacatga aacttcccat agatatgcac cctaggatct ctcaactcat 7920tccgcacaga ggccaagaaa actgctacct ccaccgggga ccagccgaga acccttgtac 7980aaagcgcaag agcatacgac tcaagactca taaccatgac ttcgcgatgg aaccgaccgg 8040cttcttttaa tcgcttttct ttagcccagg gacccatcgg gaactacaga aattaagtta 8100atacccgata gattaacgga gtattaaacc agagggagag cgaaagtaga aaaaacctac 8160cttgtagatc ttttccttca catcggtgaa ccctgcgtct atcatatttc tctgatggtc 8220ggccgccacc ttcattggcc ggttaaacct cgagcttgcc tcaatcaaca gtctgaacca 8280ctcgcccagt gagggcgcca gatcgagtgt gtcatcgtca gagaaatgtt ccatctcaaa 8340gtcggatagt tccaaccatc cacctggctt catgtgtgaa tatgcctggc gcagcagttt 8400cggccagtcg gcgatcgaac cggcaaggtc gcatgcacgg atgaagtcga agtaatctcg 8460tttgaaagtc cagtcggctt cgacgtcgtc gacgaagaat tcgaggttcg gagggaccca 8520ggcgggttgg atggggctga gatcggtgcc gattactcgg gcgttggggt gtgtgtctgc 8580gaagtcgatg gcccagatac ctttattatt atttggtcag ccatgagcga gcgggtttcc 8640tggaatagat acggcatgtg ccatacctgt tccggtgcca atgtcgaaaa cgttctcaac 8700atcctctggg atcggggcgt gaaagagatc tccactgaaa tgcagattgc gaacgtggtg 8760taataggtcc tcgcgatctt gctcttgttc gtcgtttggc agcacacact gcccttcgcg 8820gtaggagtgg tatcgacggc

cattctcgta cttgtagttt caagggcgaa ttcgcggccg 8880ctctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 8940ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 9000ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 9060tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 9120tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 9180gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 9240ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 9300tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 9360agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 9420atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 9480acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 9540actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 9600tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 9660tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 9720tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 9780tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 9840caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 9900cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 9960agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 10020acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 10080gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 10140ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 10200tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 10260ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 10320tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 10380attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 10440agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 10500ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 10560ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 10620cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 10680gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 10740tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 10800tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 10860tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta 10920tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc 10980agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc 11040agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc 11100agattgtact gagagtgcac catatcgacg ctctccctta tgcgactcct gcattaggaa 11160gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa 11220ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca 11280agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata 11340ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag 11400gatctggcta gcgatgaccc tgctgattgg ttcgctgacc atttccgggg tgcggaacgg 11460cgttaccaga aactcagaag gttcgtccaa ccaaaccgac tctgacggca gtttacgaga 11520gagatgatag ggtctgcttc agtaagccag atgctacaca attaggcttg tacatattgt 11580cgttagaacg cggctacaat taatacataa ccttatgtat catacacata cgatttaggt 11640gacactata 11649297616DNAArtificial Sequencecomplete DNA sequence of plasmid pMT3431 29ggacgcacca tccaattaga agcagcaaag cgaaacagcc caagaaaaag gtcggcccgt 60cggccttttc tgcaacgctg atcacgggca gcgatccaac caacaccctc cagagtgact 120aggggcggaa atttatcggg attaatttcc actcaaccac aaatcacagt cgtccccggt 180aatttaacgg ctgcagacgg caatttaacg gcttctgcga atcgcttgga ttccccgccc 240ctggccgtag agcttaaagt atgtcccttg tcgatgcgat gtatgaattc atggtgtttt 300gatcatttta aatttttata tggcgggtgg tgggcaactc gcttgcgcgg gcaactcgct 360taccgattac gttagggctg atatttacgt aaaaatcgtc aagggatgca agaccaaacc 420gttaaatttc cggagtcaac agcatccaag cccaagtcct tcacggagaa accccagcgt 480ccacatcacg agcgaaggac cacctctagg catcggacgc accatccaat tagaagcagc 540aaagcgaaac agcccaagaa aaaggtcggc ccgtcggcct tttctgcaac gctgatcacg 600ggcagcgatc caaccaacac cctccagagt gactaggggc ggaaatttat cgggattaat 660ttccactcaa ccacaaatca cagtcgtccc cggtaattta acggctgcag acggcaattt 720aacggcttct gcgaatcgct tggattcccc gcccctggcc gtagagctta aagtatgtcc 780cttgtcgatg cgatgtatca caacatataa atactggcaa gggatgccat gcttggagtt 840tccaactcaa tttacctcta tccacacttc tcttccttcc tcaatcctct atatacacaa 900ctggggatcc accatgagga gctcccttgt gctgttcttt gtctctgcgt ggacggcctt 960ggctagccct attcgtcctc gaccggtctc gcaggatctg tttaaccagt tcaatctctt 1020tgcacagtat tcagctgccg catactgcgg aaaaaacaat gatgccccag caggtacaaa 1080cattacgtgc acgggaaatg cctgccccga ggtagagaag gcggatgcaa cgtttctcta 1140ctcgtttgaa gactctggag tgggcgatgt caccggcttc cttgctctcg acaacacgaa 1200caaattgatc gtcctctctt tccgtggctc tcgttccata gagaactgga tcgggaatct 1260taacttcgac ttgaaaaaaa taaatgacat ttgctccggc tgcaggggac atgacggctt 1320cacttcgtcc tggaggtctg tagccgatac gttaaggcag aaggtggagg atgctgtgag 1380ggagcatccc aactatcgcg tggtgtttac cggacatagc ttgggtggtg cattggcaac 1440tgttgccgga gcagacctgc gtggaaatgg gtatgatatc gacgtgtttt catatggcgc 1500cccccgagtc ggaaacaggg cttttgcaga attcctgacc gtacagaccg gcggaacact 1560ctaccgcatt acccacacca atgatattgt ccctagactc ccgccgcgcg aattcggtta 1620cagccattct agcccagaat actggatcaa atctggaacc cttgtccccg tcacccgaaa 1680cgatatcgtg aagatagaag gcatcgatgc caccggcggc aataaccggc cgaacattcc 1740ggatatccct gcgcacctat ggtacttcgg gttaattggg acatgtcttt agtgcgcggc 1800gcggctgggt cgactctagc gagctcgaga tctagagggt gactgacacc tggcggtaga 1860caatcaatcc atttcgctat agttaaagga tggggatgag ggcaattggt tatatgatca 1920tgtatgtagt gggtgtgcat aatagtagtg aaatggaagc caagtcatgt gattgtaatc 1980gaccgacgga attgaggata tccggaaata cagacaccgt gaaagccatg gtctttcctt 2040cgtgtagaag accagacaga cagtccctga tttacccttg cacaaagcac tagaaaatta 2100gcattccatc cttctctgct tgctctgctg atatcactgt cattcaatgc atagccatga 2160gctcatctta gatccaagca cgtaattcca tagccgaggt ccacagtgga gcagcaacat 2220tccccatcat tgctttcccc aggggcctcc caacgactaa atcaagagta tatctctacc 2280gtccaataga tcgtcttcgc ttcaaaatct ttgacaattc caagagggtc cccatccatc 2340aaacccagtt caataatagc cgagatgcat ggtggagtca attaggcagt attgctggaa 2400tgtcggggcc agttggccgg gtggtcattg gccgcctgtg atgccatctg ccactaaatc 2460cgatcattga tccaccgccc acgaggcgcg tctttgcttt ttgcgcggcg tccaggttca 2520actctctcct ctagactgga aacgcaaccc tgaagggatt cttcctttga gagatggaag 2580cgtgtcatat ctcttcggtt ctacggcagg tttttttctg ctctttcgta gcatggcatg 2640gtcacttcag cgcttattta cagttgctgg tattgatttc ttgtgcaaat tgctatctga 2700cacttattag ctatggagtc accacatttc ccagcaactt ccccacttcc tctgcaatcg 2760ccaacgtcct ctcttcactg agtctccgtc cgataacctg cactgcaacc ggtgccccat 2820ggtacgcctc cggatcatac tcttcctgca cgagggcatc aagctcacta accgccttga 2880aactctcatt cttcttatcg atgttcttat ccgcaaaggt aaccggaaca accacgctcg 2940tgaaatccag caggttgatc acagaggcat acccatagta ccggaactgg tcatgccgta 3000ccgcagcggt aggcgtaatc ggcgcgatga tggcgtccag ttccttcccg gccttttctt 3060cagcctcccg ccatttctca aggtactcca tctggtaatt ccacttctgg agatgcgtgt 3120cccagagctc gttcatgtta acagctttga tgttcgggtt cagtaggtct ttgatatttg 3180gaatcgccgg ctcgccggat gcactgatat cgcgcattac gtcggcgctg ccgtcagccg 3240cgtagatatg ggagatgaga tcgtggccga aatcgtgctt gtatggcgtc cacggggtca 3300cggtgtgacc ggctttggcg agtgcggcga cggtggtttc cacgccgcgc aggataggag 3360ggtgtggaag gacattgccg tcgaagttgt agtagccgat attgagcccg ccgttcttga 3420tcttggaggc aataatgtcc gactcggact ggcgccaggg catggggatg accttggagt 3480cgtatttcca tggctcctga ccgaggacgg atttggtgaa gaggcggagg tctaacatac 3540ttcatcagtg actgccggtc tcgtatatag tataaaaagc aagaaaggag gacagtggag 3600gcctggtata gagcaggaaa agaaggaaga ggcgaaggac tcaccctcaa cagagtgcgt 3660aatcggcccg acaacgctgt gcaccgtctc ctgaccctcc atgctgttcg ccatctttgc 3720atacggcagc cgcccatgac tcggccttag accgtacagg aagttgaacg cggccggcac 3780tcgaatcgag ccaccgatat ccgttcctac accgatgacg ccaccacgaa tcccaacgat 3840cgcaccctca ccaccagaac tgccgccgca cgaccagttc ttgttgcgtg ggttgacggt 3900gcgcccgatg atgttgttga ctgtctcgca gaccatcagg gtctgcggga cagaggtctt 3960gacgtagaag acggcaccgg ctttgcggag catggttgtc agaaccgagt ccccttcgtc 4020gtacttgttt agccatgaga tgtagcccat tgatgtttcg tagccctggt ggcatatgtt 4080agctgacaaa aagggacatc taacgactta ggggcaacgg tgtaccttga ctcgaagctg 4140gtctttgaga gagatgggga ggccatggag tggaccaacg ggtctcttgt gctttgcgta 4200gtattcatcg agttcccttg cctgcgcgag agcggcgtca gggaagaact cgtgggcgca 4260gtttgtctgc acagaagcca gcgtcagctt gatagtccca taaggtggcg ttgttacatc 4320tccctgagag gtagagggga ccctactaac tgctgggcga ttgctgcccg tttacagaat 4380gctagcgtaa cttccaccga ggtcaactct ccggccgcca gcttggacac aagatctgca 4440gcggaggcct ctgtgatctt cagttcggcc tctgaaagga tcaccgattt ctttgggaaa 4500tcaataacgc tgtcttccgc aggcagcgtc tggactttcc attcatcagg gatggttttt 4560gcgaggcggg cgcgcttatc agcggccagt tcttcccagg attgaggcat tctgtgttag 4620cttatagtca ggatgttggc tcgacgagtg taaactggga gttggcatga gggttatgta 4680ggcttcttta gccccgcatc cccctcattc tcctcattga tcccggggga gcggatggtg 4740ttgataagag actaattata gggtttagct ggtgcctagc tggtgattgg ctggcttcgc 4800cgaattttac gggccaagga aagctgcaga accgcggcac tggtaaacgg taattaagct 4860atcagcccca tgctaacgag tttaaattac gtgtattgct gataaacacc aacagagctt 4920tactgaaaga tgggagtcac ggtgtggctt ccccactgcg attattgcac aagcagcgag 4980ggcgaacttg actgtcgtcg ctgagcagcc tgcagtcaaa catacatata tatcaaccgc 5040gaagacgtct ggccttgtag aacacgacgc tccctagcaa cacctgccgt gtcagcctct 5100acggttgtta cttgcattca ggatgctctc cagcgggcga gctattcaaa atattcaaag 5160caggtatctc gtattgccag gattcagctg aagcaacagg tgccaaggaa atctgcgtcg 5220gttctcatct gggcttgctc ggtcctggcg tagatctaga gtcgacctgc aggcatgcgg 5280cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 5340acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 5400cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 5460attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 5520cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 5580caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 5640caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 5700ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 5760cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 5820ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 5880tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 5940gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 6000ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 6060ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 6120gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 6180aaagagttgg tagctcttga tccgacaaac aaaccaccgc tggtagcggt ggtttttttg 6240tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 6300ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 6360tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 6420aaagtatata tgagtaaact tggtctgaca gttaagcaag gattttctta acttcttcgg 6480cgacagcatc accgacttcg gtggtactgt tggaaccacc taaatcacca gttctgatac 6540ctgcatccaa aaccttttta actgcatctt caatggcctt accttcttca ggcaagttca 6600atgacaattt caacatcatt gcagcagaca agatagtggc gatagggttg accttattct 6660ttggcaaatc tggagcagaa ccgtggcatg gttcgtacaa accaaatgcg gtgttcttgt 6720ctggcaaaga ggccaaggac gcagatggca acaaacccaa ggaacctggg ataacggagg 6780cttcatcgga gatgatatca ccaaacatgt tgctggtgat tataatacca tttaggtggg 6840ttgggttctt aactaggatc atggcggcag aatcaatcaa ttgatgttga accttcaatg 6900tagggaattc gttcttgatg gtttcctcca cagtttttct ccataatctt gaagaggcca 6960aaacattagc tttatccaag gaccaaatag gcaatggtgg ctcatgttgt agggccatga 7020aagcggccat tcttgtgatt ctttgcactt ctggaacggt gtattgttca ctatcccaag 7080cgacaccatc accatcgtct tcctttctct taccaaagta aatacctccc actaattctc 7140tgacaacaac gaagtcagta cctttagcaa attgtggctt gattggagat aagtctaaaa 7200gagagtcgga tgcaaagtta catggtctta agttggcgta caattgaagt tctttacgga 7260tttttagtaa accttgttca ggtctaacac tgccggtacc ccatttagga ccacccacag 7320cacctaacaa aacggcatca gccttcttgg aggcttccag cgcctcatct ggaagtggaa 7380cacctgtagc atcgatagca gcaccaccaa ttaaatgatt ttcgaaatcg aacttgacat 7440tggaacgaac atcagaaata gctttaagaa ccttaatggc ttcggctgtg atttcttgac 7500caacgtggtc acctggcaaa acgacgatct tcttaggggc agacatactc ttcctttttc 7560aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaa 76163020DNAArtificial SequencePCR primer 30ggtaaccccc tatcacaagc 203122DNAArtificial SequencePCR primer 31cgatcgactt gaaaaccatg tc 223223DNAArtificial SequencePCR primer 32gaaactacaa gtacgagaat ggc 233344DNAArtificial SequencePCR primer 33gcttgtgata gggggttacc actcattgca tttcagtagt aacc 443418DNAArtificial SequencePCR primer 34gttacgccac gtccctgc 183521DNAArtificial SequencePCR primer 35ccccagtcag tacatgcttc g 213625DNAArtificial SequencePCR primer 36gatctacaag gtaggttttt tctac 253729DNAArtificial SequencePCR primer 37ggatcccttt tcatttaatc taactaagg 29

Claims

1. An isolated polypeptide, selected from the group consisting of:(a) a polypeptide comprising an amino acid sequence having at least 66%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide of SEQ ID NO: 2;(b) a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions, even more preferably at least medium-high stringency conditions, and most preferably at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) cDNA sequence contained in the DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii);(c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide coding sequence of SEQ ID NO: 1;(d) a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NO: 2.

2. The polypeptide of claim 1, comprising or consisting of the amino acid sequence of SEQ ID NO: 2.

3. The polypeptide of claim 2, comprising or consisting of the mature polypeptide of SEQ ID NO: 2.

4. The polypeptide of claim 1, which is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 1.

5. The polypeptide of claim 4, which is encoded by a polynucleotide comprising or consisting of the mature polypeptide coding sequence of SEQ ID NO: 1.

6. The polypeptide of claim 1, wherein the mature polypeptide is amino acids 20 to 79 of SEQ ID NO: 2.

7. The polypeptide of claim 1, wherein the mature polypeptide coding sequence is nucleotides 58 to 237 of SEQ ID NO: 1.

8. An isolated polynucleotide comprising a nucleotide sequence that encodes the polypeptide of claim 1.

9. A nucleic acid construct comprising the polynucleotide of claim 8 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

10. A recombinant expression vector comprising the nucleic acid construct of claim 9.

11. A recombinant host cell comprising the nucleic acid construct of claim 9.

12. A method of producing the polypeptide of claim 1, comprising: (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

13. A method of producing the polypeptide of claim 1, comprising: (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

14. A mutant fungal host, wherein the mutant has a reduced or no expression of an endogenous polypeptide selected from the group consisiting of:(a) a polypeptide comprising an amino acid sequence having at least 66%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide of SEQ ID NO: 2;(b) a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions, even more preferably at least medium-high stringency conditions, and most preferably at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) cDNA sequence contained in the DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii);(c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide coding sequence of SEQ ID NO: 1;(d) a polypeptide comprising the motif: EXXCGXXGXMXXDPXXLPEGVXXXXXRXCA; and(e) a variant comprising a substitution, deletion, and/or insertion of one or several amino acids of the mature polypeptide of SEQ ID NO: 2.

15. The mutant according to claim 14, wherein the endogenous polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 2; or a fragment thereof.

16. The mutant according to claim 15, wherein the endogenous polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 2.

17. The mutant according to claim 16, wherein the endogenous polypeptide is encoded by a polynucleotide which comprises or consists of the nucleotide sequence of SEQ ID NO: 1; or a subsequence thereof.

18. The mutant according to claim 17, wherein the endogenous polypeptide is encoded by a polynucleotide which comprises or consists of the mature polypeptide coding sequence of SEQ ID NO: 1.

19. The mutant according to claim 15, wherein the mature endogenous polypeptide is amino acids 20 to 79 of SEQ ID NO: 2.

20. The mutant according to claim 14, wherein the mature endogenous polypeptide coding sequence is nucleotides 58 to 237 of SEQ ID NO: 1.

21. The mutant fungal host according to claim 1, wherein the fungal cell is a filamentous fungal cell or a yeast cell.

22. The mutant fungal host according to claim 21, wherein the filamentous fungal host is selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

23. The mutant fungal host according to claim 22, wherein the Aspergillus cell is selected from Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae.

24. The mutant cell of claim 1, further comprising a gene encoding a native or heterologous protein.

25. A method of producing a protein of interest comprising: (a) cultivating a mutant cell under conditions conducive for production of the protein wherein the mutant has a reduced or no expression of an endogenous polypeptide compared to a parent host cell grown under the same conditions, and wherein the expression of said endogenous polypeptide commits the cell to apoptosis; and optionally (b) recovering the protein of interest.

26. A method of producing a protein of interest, comprising: (a) cultivating the mutant cell of claim 24 under conditions conducive for production of the protein; and optionally (b) recovering the protein.

27. The method according to claim 25, wherein the protein is a heterologous protein.

28. The method according to claim 25, wherein the reduced expression or no expression of the endogenous polypeptide is provided by random mutation, site specific mutation, deletion, or double-stranded inhibitory RNA, by targeting the coding sequence or a control element operably linked to the coding sequence of the endogenous polypeptide.

29. The method according to claim 25, wherein elimination of the endogenous polypeptide is provided by a deletion of either at least a part of the gene encoding the endogenous polypeptide or the promoter controlling expression of the said gene.

30. The method according to claim 25, wherein the filamentous fungal host cell is selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

31. The method according to claim 30, wherein the Aspergillus host cell is selected from the group consisting of Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cells.

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